OPTICAL METHODS.

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.

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.:

S = C - 0.4891A
0.810
and R = A - S
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].

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.

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.

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

[α]°D = - 29.68 × 100= -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]D⁸⁸ ° = - 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.

Then L = R÷ 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 used26.048 grams.
First polarization88°.5
Polarization after inversion 10°.5
Temperature20°.0
Percentage of sucrose58.4
Rotation due to dextrose30°.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.

Polarimetricreadingfromthe100cubiccentimeterflask,20°.84
””””200”””10°.15

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:

a =300milligrams = 163.8milligramsinvert sugar.
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:

Sucrose101.00milligrams.
Invert sugar  139.46”
Dextrose19.92”
Sum260.38”

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:

Copperobtainedbeforeinversion= a =150milligrams.
””after”= b =250”
”””destroying lev’e= d =137.5”
”equivalenttoinverted sucrose= b - a = c =100”
”””levulose= b - d = e =112.5”
a =150milligramsCu= 44.0milligramsinvert sugar
45.3”dextrose
42.5”levulose.
d =137.5””= 41.55”dextrose.
c =100””= 29.5”invert sugar
= 28.025sucrose.
e =112.5””= 31.9”levulose.
14.75milligramsofdextrose =48.5milligramsCu.
14.75””levulose =51.5””

137.5 - 48.5 = 89.0 milligrams Cu due to dextrose present before inversion.

150.0 -89.0milligramsCu= 27milligramsdextrose before inversion.
89.0””= 61.0”Cu due to levulose present before inversion.
61.0””= 17.8”levulose before inversion.
17.8 × 2 =35.6milligramsinvert sugarpresentbeforeinversion.
27.0 - 17.8 = 9.2”dextrose”””

Again:

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

Sucrose= 28.025milligrams.
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.

252. Separation of Sugars by Lead Oxid.—In addition to the combination with the earthy bases, sugar forms well defined compounds with lead oxid. One of these compounds is of such a nature as to have considerable analytical and technical value. Its composition and the method of preparing it have been pointed out by Kassner.[207]

Sucrose, under conditions to be described, forms with the lead oxid a diplumbic saccharate, which separates in spheroidal crystals, and has the composition corresponding to the formula C₁₂H₁₈O₁₁Pb₂ + 5H₂O. The precipitation takes place quantitively and should be conducted as follows:

The substance containing the sucrose, which may be molasses, sirups or concentrated juices, is diluted with enough water to make a sirup which is not too viscous. Lead oxid suspended in water is stirred into the mass in such proportion as to give about two parts of oxid to one of the sugar. The stirring is continued for some time until the oxid is thoroughly distributed throughout the mass and until it becomes thick by the commencement of the formation of the saccharate. As soon as the mass is sufficiently thickened to prevent the remaining lead oxid from settling, the stirring may be discontinued and the mixture is left for twenty-four hours, at the end of which time the sucrose has all crystallized in the form of lead saccharate. The crystals of lead saccharate can be separated by a centrifugal machine or by passing through a filter press, and are thoroughly washed with cold water, in which they are almost insoluble. The washed crystals are beaten up with water into a thick paste and the lead separated as basic carbonate by carbon dioxid. The sucrose is found in solution in the residual liquor and is concentrated and crystallized in the usual way.

Reducing sugars have a stronger affinity for the lead oxid than the sucrose, and this fact is made use of to effect a nearly complete separation when they are mixed together. In order to secure this the lead oxid is added in the first place only in sufficient quantity to combine with the reducing sugars present, the process being essentially that described above. The reducing sugars which are precipitated as lead dextrosates, lead levulosates, etc., are separated in the usual way by a centrifugal or a filter press, and the resulting liquor, which contains still nearly all the sucrose, is subjected to a second precipitation by the addition of lead oxid. The second precipitation obtained is almost pure diplumbic saccharate.

In the precipitation of the sugar which is contained in the beet molasses, where only a trace or very little invert sugar is present, the sucrose is almost quantitively separated, and by the concentration of the residual liquor, potash salts are easily obtained. In this case, after the decomposition of the lead saccharate by carbon dioxid, the residual sugar solution is found entirely free of lead. Where invert sugar is present, however, in any considerable proportions, it is found to exercise a slightly soluble influence on the lead saccharate, and in this case a trace of lead may pass into solution. For technical purposes, this is afterwards separated by hydrosulfuric acid or the introduction of lime sulfid.

Lead oxid is regenerated from the basic lead carbonate obtained by heating in retorts to a little above 260°, and the carbon dioxid evolved can also be used again in the technical process.

253. Commercial Glucose and Grape Sugar.—The commercial products obtained by the hydrolysis of starch are known in the trade as glucose or grape sugar. The former term is applied to the thick sirup obtained by concentrating the products of a partial hydrolysis, while the latter is applied to the solid semi-crystalline mass, secured by continuing the hydrolyzing action until the intermediate products are almost completely changed to dextrose. In this country the starch employed is obtained almost exclusively from maize, and the hydrolyzing agent used is sulfuric acid.[208] The products of conversion in glucose are chiefly dextrins and dextrose with some maltose, and in grape sugar almost entirely dextrose. When diastase is substituted for an acid, as the hydrolytic agent, maltose is the chief product, the ferment having no power of producing dextrose. In the glucose of Japan, known as midzu ame dextrin and maltose are the chief constituents.[209]

Commercial glucose is used chiefly by confectioners for manufacturing table sirups and for adulterating honey and molasses.

Commercial grape sugar is chiefly employed by brewers as a substitute for barley and other grains.

In Europe, the starch which is converted into glucose, is derived principally from potatoes. The method employed in conversion, whether an acid or diastatic action, is revealed not only by the nature of the product, but also by the composition of its ash. In the case of diastatic conversion the ash of the sample will contain only a trace of sulfates, no chlorin, and be strongly alkaline, while the product of conversion with sulfuric acid will give an ash rich in sulfates with a little lime and be less strongly alkaline.

The process of manufacture in this country consists in treating the starch, beaten to a cream with water, with sulfuric acid, usually under pressure, until the product shows no blue color with iodin. The excess of acid is removed with marble dust, the sirup separated by filtration, whitened by bleaching with sulfurous acid or by passing it through bone-black and evaporated to the proper consistence in a vacuum. The solid sugar, consisting mostly of dextrose, is made in the same manner, save that the heating with the acid is continued until the dextrin and maltose are changed into maltose. The product is either obtained in its ordinary hydrated form or by a special method of crystallization secured as bright anhydrous crystals. Solutions of dextrose, when first made, show birotation, but attain their normal gyrodynatic state on standing for twenty-four hours in the cold, or immediately on boiling.

254. Methods Of Separation.—The accurate determination of the quantities of the several optically active bodies formed in commercial glucose is not possible by any of the methods now known. Approximately accurate data may be secured by a large number of processes, and these are based chiefly on the ascertainment of the rotation and reducing power of the mixed sugars, the subsequent removal of the dextrose and maltose by fermentation or oxidation and the final polarization of the residue. The difficulties which attend these processes are alike in all cases. Fermentation may not entirely remove the reducing sugars or may act slightly on the dextrin. In like manner the oxidation of these sugars by metallic salts may not entirely decompose them, may leave an optically active residue, or may affect the optical activity of the residual dextrin. The quantitive methods of separating these sugars by means of phenylhydrazin, lead salts or earthy bases have not been developed into reliable and applicable laboratory processes. At the present time the analyst must be contented with processes confessedly imperfect, but which, with proper precautions, yield data which are nearly correct. The leading methods depending on fermentation and oxidation combined with polarimetric observations will be described in the subjoined paragraphs.

255. Fermentation Method.—This process is based on the assumption that, under certain conditions, dextrose and maltose may be removed from a solution and the dextrin be left unchanged. In practice, approximately accurate results are obtained by this method, although the assumed conditions are not strictly realized. In the prosecution of this method the polarimetric reading of the mixed sugars is made, and the maltose and dextrose removed therefrom by fermentation with compressed yeast. The residual dextrins are determined by the polariscope on the assumption that their average gyrodynat is 193. In the calculation of the quantities of dextrose and maltose their gyrodynats are fixed at 53 and 138 respectively. The total quantity of reducing sugar is determined by the usual processes. The relative reducing powers of dextrose and maltose are represented by 100 and 62 respectively. The calculations are made by the following formulas:[210]

Whence R= d + 0.62m(1)
P= 53d + 138m + 163dʹ (2)
Pʹ= 193dʹ(3)

From these three equations the values of d, m, and dʹ are readily calculated:

Example: To find d and m:

Subtract (3) from (2) P =53d + 133m + 193dʹ
Pʹ =193dʹ
P - Pʹ =53d + 138m  (4)
Multiply (1) by 53 and subtract from (4) P - Pʹ =53d + 138m
53R =53d + 32.86m
P - Pʹ -53R = 105.14m  (5)
Whence m = P - Pʹ - 53R (6)
105.14

d = R - 0.62m(7)

dʹ = Pʹ(8)
193

Sidersky assigns the values [a]D = 138.3 and [a]D = 194.8 to maltose and dextrin respectively in the above formulas.[211]

Illustration: In the examination of a sample 26.048 grams of midzu ame in 100 cubic centimeters were polarized in a 200 millimeter tube and the following data were obtained:

Polarization of sample in angular degrees 69°.06, which is equal to an apparent gyrodynat of 132.6:

Total reducing sugar as dextrose 33.33 per cent:

Polarization in angular degrees after fermentation 30°.84 = [a]D = 59.2.

Substituting these values in the several equations gives the following numbers:

Summary: Sample of midzu ame:

Percentageofdextrin30.67percent.
””maltose53.01””
””dextrose00.47””
””water14.61””
””ash00.31””
Sum99.07””
Undetermined  0.93””

For polarization the lamplight shadow polariscope employed for sugar may be used, and the degrees of the sugar (ventzke) scale converted into angular degrees by multiplying by 0.3467.

The process of fermentation is conducted as described in the paragraph given further on, relating to the determination of lactose in the presence of sucrose.

256. The Oxidation Method.—The removal of the reducing sugars may be accomplished by oxidation instead of fermentation. The process of analysis is in all respects similar to that described in the foregoing paragraph, substituting oxidation for fermentation.[212] For the oxidizing agent mercuric cyanid is preferred, and it is conveniently prepared by dissolving 120 grams of mercuric cyanid and an equal quantity of sodium hydroxid in water mixing the solutions and completing the volume to one liter. If a precipitate be formed in mixing the solutions it should be removed by filtering through asbestos. For the polarization, ten grams of the sugars in 100 cubic centimeters is a convenient quantity. Ten cubic centimeters of this solution are placed in a flask of water marked at fifty cubic centimeters, a sufficient quantity of the mercuric cyanid added to remain in slight excess after the oxidation is finished (from twenty to twenty-five cubic centimeters) and the mixture heated to the boiling point for three minutes. The alkali, after cooling, is neutralized with strong hydrochloric acid and the passing from alkalinity to acidity will be indicated by a discharge of the brown color which is produced by heating with the alkaline mercuric cyanid. The heating with the mercury salt should be conducted in a well ventilated fume chamber.

The calculation of the results is conducted by means of the formulas given in the preceding paragraph. In the original paper describing this method, it was stated that its accuracy depended on the complete oxidation of the reducing sugar in a manner leaving no optically active products, and on the inactivity of the reagents used in respect to the dextrin present. These two conditions are not rigidly fulfilled, as is shown by Wilson.[213] According to his data maltose leaves an optically active residue, which gives a somewhat greater right hand rotation than is compensated for by the diminished rotation of the dextrin. Wilson, however, confesses that the dextrin used contained reducing sugars, which would not be the case had it been prepared by the process of treating it with alkaline mercuric cyanid as above indicated. Upon the whole, the oxidation of the reducing sugar by a mercury salt gives results which, while not strictly accurate, are probably as reliable as those afforded by fermentation. The author has attempted to supplant both the oxidation and fermentation methods by removing the reducing sugars with a precipitating reagent, such as phenylhydrazin, but the methods are not sufficiently developed for publication.

257. Removal of Dextrose by Copper Acetate.—Maercker first called attention to the fact that Barfoed’s reagent (one part copper acetate in fifteen parts of water, and 200 cubic centimeters of this solution mixed with five cubic centimeters of thirty-eight per cent. acetic acid) reacts readily with dextrose, while it is indifferent to maltose and dextrins. Sieben’s method of removing dextrose is based on this fact.[214] It is found that under certain conditions pure maltose does not reduce either the acidified or neutral solution of copper acetate, while dextrose or a mixture of dextrose and maltose does so readily. It is also shown that the fermentation residue under suitable conditions acts like maltose. Maltose solutions reduce the reagent after boiling four minutes while at 40°-45° they have no effect even after standing four days. The amount of copper deposited by dextrose, under the latter conditions, is found to depend to a certain extent on the amount of free acetic acid present, and as the solutions of copper acetate always contain varying quantities of acetic acid which cannot be removed without decomposition and precipitation of basic salt, the use of an absolutely neutral solution is impracticable. The reagent prepared according to Barfoed’s directions is almost saturated, but a half normal solution is preferable. Sieben proposes two solutions: I, containing 15.86 grams copper and 0.56 gram acetic anhydrid per liter; II, containing 15.86 grams copper and three grams acetic anhydrid per liter. The reduction of the dextrose is secured by placing 100 cubic centimeters of the solution in a bottle, adding the sugar solution, stoppering and keeping in a water-bath at 40°-45° two or three days. An aliquot portion is then drawn off and the residual copper precipitated by boiling with forty-five cubic centimeters of the alkali solution of the fehling reagent and forty cubic centimeters of one per cent dextrose solution, filtered and weighed as usual. The results show that either solution can be used, and that standing for two days at 45° is sufficient. One hundred cubic centimeters of the copper solution are mixed with ten cubic centimeters of the sugar solution containing from two-tenths to five-tenths gram of dextrose, as this dilution gives the best results. No reduction is found to have taken place when solutions containing five-tenths gram maltose or five-tenths gram fermentation residue are used. The data can not be compiled in the form of a table similar to Allihn’s, as it is impossible to obtain a solution of uniform acidity each time, and the solution will have to be standardized by means of a known pure dextrose solution and the result obtained with the unknown sugar solution properly diluted compared with this. This method of Sieben’s has never been practiced to any extent in analytical separations and can not, therefore, be strongly recommended without additional experience.

258. Removal of Dextrin by Alcohol.—By reason of its less solubility, dextrin can be removed from a solution containing also dextrose and maltose by precipitation with alcohol. It is impracticable, however, to secure always that degree of alcoholic concentration which will cause the coagulation of all the dextrins without attacking the concomitant reducing sugars. In this laboratory it has been found impossible to prepare a dextrin by alcoholic precipitation, which did not contain bodies capable of oxidizing alkaline copper solutions.

The solution containing the dextrin is brought to a sirupy consistence by evaporation and treated with about ten volumes of ninety per cent alcohol. After thorough mixing, the precipitated dextrin is collected on a filter and well washed with alcohol of the strength noted. It is then dried and weighed. If weaker solutions of dextrin are used, the alcohol must be of correspondingly greater strength. In the filtrate the residual maltose and dextrose may be separated and determined by the chemical and optical methods already described.