Part 26

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:

66.4: 52.5 = _x_: 26.048

Whence _x_ = 32.74.

In the one case three times the normal weight of milk is 61.53 and in the other twice the normal weight, 65.48 grams.

=244. Error due to Volume of Precipitate.=—Vieth states that the volume allowed for the precipitated solids in the original process, _viz._, two and four-tenths cubic centimeters, is not sufficiently large.[202] In such cases it is quite difficult to decide on any arbitrary correction based on the supposed quantities of fat and albuminoids present. A better method than to try to compensate for any arbitrary volume is to remove entirely the disturbing cause or eliminate it by indirect means. To wash the precipitate free of sugar without increasing the bulk of the filtrate unduly would be extremely difficult and tend, moreover, to bring some of the precipitated matters again into solution. It is better, therefore, to eliminate the error by double dilution and polarization (=86=). The principle of this method is based on the fact, that, within limits not sensibly affecting the gyrodynat by reason of different densities, the polarizations of two solutions of the same substance are inversely proportional to their volumes.

For convenience, it is recommended that the volumes of the samples in each instance be 100 and 200 cubic centimeters, respectively, in which case the true reading is obtained by the simple formula given in the latter part of =86=.

In this laboratory the double dilution method of determining the volume of the precipitate is conducted as follows:[203]

In each of two flasks marked at 100 and 200 cubic centimeters, respectively, are placed 65.52 grams of milk, four cubic centimeters of mercuric nitrate added, the volume completed to the mark and the contents of the flask well shaken.

After filtering, the polarization is made in a 400 millimeter tube by means of the triple shadow polariscope described in =75=. From the reading thus obtained the volume of the precipitate and the degree of correction to be applied are calculated as in the subjoined example. The flasks should be filled at near the temperature at which the polarizations are made and the observation room must be kept at practically a constant temperature of 20° to avoid the complications which would be produced by changes in the gyrodynat of lactose and the value of the quartz plates and wedges of the apparatus by marked variations in temperature.

_Example._—Weight of milk used in each case 65.52 grams.

Polarimetric reading from the 100 cubic centimeter flask, 20°.84 ” ” ” ” 200 ” ” ” 10°.15 Then 10.15 × 2 = 20.30 20.84 - 20.30 = 0.54 0.54 × 2 = 1.08 20.84 - 1.08 = 19.76 19.76 ÷ 4 = 4.94,

which is the corrected reading showing the percentage of lactose in the sample used.

The volume of the precipitate is calculated as follows:

20.84 ÷ 4 = 5.21, the apparent percentage of lactose present.

Then 5.21: 4.94 = 100: _x_.

Whence _x_ = 94.82. From this number it is seen that the true volume of the milk solution polarized is 94.82 instead of 100 cubic centimeters, whence the volume occupied by the precipitate is 100 - 94.82 = 5.18 cubic centimeters. So little time is required to conduct the analysis by the double dilution method as to render it preferable in all cases where incontestable data are desired. Where arbitrary corrections are made the volume allowed for the precipitate may vary from two and a half cubic centimeters in milks poor in fat, to six for those with a high cream content.

For milks of average composition sufficient accuracy is secured by making an arbitrary correction of five cubic centimeters for the volume of the precipitate.

SEPARATION OF SUGARS BY CHEMICAL AND CHEMICAL-OPTICAL METHODS.

=245. Conditions of Separation.=—In the foregoing paragraphs the optical methods for determining certain sugars have been described. Many cases arise, however, in which these processes are inapplicable or insufficient. In these instances, the analyst, as a rule, will be able to solve the problem presented by the purely chemical methods which have been previously described, or by a combination of the chemical and optical processes. Not only have the different sugars distinctive relations to polarized light, but also they are oxidized by varying quantities of metallic salts and these differences are sufficiently pronounced to secure in nearly every instance, no matter how complex, data of a high degree of accuracy.

The carbohydrates of chief importance, from an agricultural point of view, are starch and sucrose; while the alternation products of chief importance, derived therefrom by chemical and biological means, are dextrin, maltose, dextrose and invert sugar.

=246. Sucrose, Levulose, and Dextrose.=—The purely chemical methods of separating these three sugars have been investigated by Wiechmann.[204] They are based on the data obtained by determining the percentage of reducing sugars, both before and after the inversion of the sucrose, and before and after the removal of the levulose. For the destruction of the levulose, the method of Sieben is employed, and attention is called to the fact that the complete removal of the levulose by this process is difficult of accomplishment, and is probably attended with alterations of the other sugars present.

=247. Sieben’s Method of Determining Levulose.=—The decomposing action of hot hydrochloric acid on levulose, and its comparative inaction on dextrose are the basis of Sieben’s process.[205] The hydrochloric acid employed should contain about 220 grams of the pure gas per liter, that is, be of twenty-two per cent strength, corresponding to 1.108 specific gravity. If the substance acted on be invert sugar, its solution should be approximately of two and a half per cent strength. To 100 cubic centimeters of such a solution, sixty of the hydrochloric acid are added, and the mixture immersed in boiling water for three hours.

After quickly cooling, the acid is neutralized with sodium hydrate of thirty-six times normal strength. Ten cubic centimeters of the hydrate solution will thus neutralize the sixty of hydrochloric acid which have been used to destroy the levulose. The work of Wiechmann discloses the fact, easily prevised, that the method used for destroying levulose is not always effective and that action of the reagent is not exclusively confined to the levogyrate constituent of the mixture. Nevertheless, data of reasonable accuracy may be secured by this process, which is best carried out as described by Wiechmann. In this connection the possibility of the polymerization of the dextrose molecules, when heated with hydrochloric acid, must not be overlooked.

=248. The Analytical Process.=—The total quantity of invert sugar in a given solution is determined by the methods already given (=136, 141=.)

After this has been accomplished, the levulose is destroyed as described above, and the dextrose determined by any approved method (=136, 140=). In the presence of sucrose, the sum of the reducing sugars is first determined as in =136, 142=. After the inversion of the sucrose, the invert sugar is again determined, and the increased quantity found, calculated to sucrose. The levulose is then destroyed by hydrochloric acid, and the dextrose determined as described above. The quantity of sucrose may also be determined by an optical method (=91, 92, 94=.).

=249. Calculation of Results.=—If we represent by a the weight of metallic copper reduced by the invert sugar present in a solution containing sucrose, and by _b_ that obtained after the inversion of the sucrose, the quantity of copper corresponding to the sucrose is _b - a_ = _c_. After the destruction of the levulose, the copper reduced by the residual dextrose may be represented by _d_. The weight of copper equivalent to the levulose is, therefore, _b - d_ = _e_. From the tables already given, the corresponding quantities of the sugars equivalent to _c, d_, and _e_ are directly taken. Example:

{ 163.8 milligrams invert sugar. _a_ = 300 milligrams = { 156.5 ” dextrose. { 185.63 ” levulose.

_b_ = 500 ”

_d_ = 275 ” = 142.8 ” dextrose.

_c_ = 200 ” = 106.3 ” invert sugar.

_e_ = 225 ” = 133.89 ” levulose.

The 106.3 milligrams of invert sugar equivalent to _c_, correspond to 101 milligrams of sucrose. The quantity of dextrose equivalent to 275 milligrams of copper is 142.8. Of this amount 53.15 milligrams are due to the inverted sucrose, leaving 89.65 milligrams arising from the invert sugar and dextrose originally present. This quantity is equivalent to 175 milligrams of copper.

Of the 300 milligrams of copper obtained in the first instance, 125 are due to levulose in the original sample, corresponding to 69.73 milligrams which number, multiplied by two, gives the invert sugar present.

The sample examined, therefore, had the following composition:

Sucrose 101.00 milligrams. Invert sugar 139.46 ” Dextrose 19.92 ” ------ Sum 260.38 ”

On the other hand, if the invert sugar be calculated from the quantity corresponding to the 225 milligrams of copper corresponding to _e_, the data will be very different from those given above. In this instance of the levulose found corresponding to 225 milligrams of copper, _viz._, 133.89, 53.15 milligrams are due to the inverted sucrose. Then the quantity due to the invert sugar at first present is 133.89 - 53.15 = 80.74 milligrams. Since half the weight of invert sugar is levulose, the total weight of the invert sugar at first present is 161.48, leaving only 8.91 milligrams due to added dextrose. The difficulties in these calculations doubtless arise from the imperfect destruction of the levulose, and from variations in the reducing action of sugars on copper salts in the presence of such large quantities of sodium chlorid.

=250. Calculation from Data obtained with Copper Carbonate.=—The wide variations observed in different methods of calculations in the preceding paragraph, are due in part to the different degrees of oxidation exerted on alkaline copper tartrate by the dextrose and levulose. Better results are obtained by conducting the analytical work with Ost’s modification of Soldaini’s solution (=128=).

The relative quantities of levulose and dextrose oxidized by this solution are almost identical, and the calculations, therefore, result in nearly the same data, whether made from the numbers obtained with the residual dextrose or from the levulose destroyed. The method of applying this method is illustrated in the following calculation.

_Example._—In a mixture of sucrose, invert sugar, and dextrose, the quantities of copper obtained by using the copper carbonate solution were as follows:

Copper obtained before inversion = _a_ = 150 milligrams. ” ” after ” = _b_ = 250 ” ” ” ” destroying lev’e = _d_ = 137.5 ” ” equivalent to inverted sucrose = _b_ - _a_ = _c_ = 100 ” ” ” ” levulose = _b_ - _d_ = _e_ = 112.5 ”

{44.0 milligrams invert sugar. _a_ = 150 milligrams Cu = {45.3 ” dextrose. {42.5 ” levulose. _d_ = 137.5 ” ” = 41.55 ” dextrose. _c_ = 100 ” ” = 29.5 ” invert sugar = 28.025 sucrose. _e_ = 112.5 ” ” = 31.9 ” levulose.

14.75 milligrams of dextrose = 48.5 milligrams Cu. 14.75 ” ” levulose = 51.5 ” ”

137.5 - 48.5 = 89.0 milligrams Cu due to dextrose present before inversion. 89.0 milligrams Cu = 27 milligrams dextrose before inversion. 150.0 - 89.0 ” ” = 61.0 ” Cu due to levulose present before inversion. 61.0 ” ” = 17.8 ” levulose before inversion. 17.8 × 2 = 35.6 milligrams invert sugar present before inversion. 27.0 - 17.8 = 9.2 ” dextrose ” ” ”

Again:

112.5 - 51.5 = 61.0 milligrams Cu due to levulose present before inversion. 61.0 milligrams Cu = 17.8 milligrams levulose. 17.8 ” levulose indicate 35.6 milligrams invert sugar. Dextrose in invert sugar before inversion = 17.8 milligrams. Total dextrose before inversion = 27.0 milligrams. Dextrose above amount required for invert sugar = 27.0 - 17.8 = 9.2 milligrams.

The respective quantities of the three sugars in the solution are, therefore:

Sucrose = 28.025 milligrams. Invert sugar = 35.6 ” Dextrose = 9.2 ”

The calculations made from the later data (=234=) give almost the same results.

=251. Winter’s Process.=—Winter has proposed a method of separating dextrose and levulose in the presence of sucrose based on the selective precipitation produced on treating mixtures of these sugars in solution with ammoniacal lead acetate.[206]

The reagent is prepared immediately before use by adding ammonia to a solution of lead acetate until the opalescence which is at first produced just disappears. The separation is based on the fact that the compound of sucrose with the reagent is easily soluble in water, while the salts formed with levulose and dextrose are insoluble. The separation of the sugars is accomplished as follows:

The ammoniacal lead acetate is added to the solution of the mixed sugars until no further precipitate is produced. The precipitated matters are digested with a large excess of water and finally separated by filtration. The sucrose is found in the filtrate in the form of a soluble lead compound, from which it is liberated by treatment with carbon dioxid. The lead carbonate produced is separated by filtration and the sucrose is estimated in an aliquot part of the filtrate by optical or chemical methods. The precipitate containing the lead compounds of dextrose and levulose is washed free of sucrose, suspended in water and saturated with carbon dioxid. By this treatment the lead compound with dextrose is decomposed and, on filtration, the dextrose will be found in the filtrate, while the lead compound of the levulose is retained upon the filter with the lead carbonate. After well washing the precipitate, it is again suspended in water and saturated with hydrogen sulfid. By this treatment the lead levulosate compound is broken up and the levulose obtained, on subsequent filtration, in the filtrate. The dextrose and levulose, after separation as above described, may be determined in aliquot parts of their respective filtrates by the usual gravimetric methods. Before determining the levulose the solution should be heated until all excess of hydrogen sulfid is expelled.

This method was used especially by Winter in separating the various sugars obtained in the juices of sugar cane. It has not been largely adopted as a laboratory method, and on account of the time and trouble required for its conduct, is not likely to assume any very great practical importance.