THE ANALYTICAL PROCESS.
76. General Principles.—Having described the instruments chiefly employed in the optical examination of sugar solutions, the next step is to apply them to the analytical work. A common set of directions for use will be found applicable to all instruments with such modifications only as are required by peculiarities of construction. With the best made instruments it is always advisable to have some method of controlling the accuracy of the observation. The simplest way of doing this is to test the apparatus by standard quartz plates. These plates are made from right-handed polarizing quartz crystal ground into plates of definite thickness and accurately tested by standard instruments. Theoretically such quartz plates deflect the plane of polarized light in a degree proportionate to their thickness, but practically some small deviations from the rule are found. With a source of light of the same tint, and at a constant temperature, such plates become a safe test for the accuracy of the graduation of polariscopes. They are more convenient for use than pure sugar solutions of known strength which are the final standards in all disputed cases. These quartz plates are conveniently mounted in tubes of the same size as those holding the sugar solution, and thus fit accurately into the trough of the polariscope, the optical axis of which passes through their center. The quartz plate when used for setting the scale of a polariscope should be placed always in the same position. In some plates slight differences of reading may be noticed on rotating the tubes holding them. Theoretically, such differences should not exist, but in practice they are sometimes found. The temperature of observation should also be noted, and if not that at which the value of the plate was fixed a proper correction should be made.
77. Setting the Polariscope.—While mention has been made of several forms of apparatus in the preceding paragraphs, those in common use are limited to a very small number. In this country quite a number of color instruments may still be found, together with a few laurents, and a constantly increasing number of shadow instruments for use with lamp light. The following description of setting the polariscope is especially adapted to the last named instrument, but the principles of adjustment are equally applicable to all.
The scale of the instrument is first so adjusted by means of the adjusting screws provided with each instrument, as to bring the zero of the vernier and that of the scale exactly together. The telescope or ocular is then adjusted until the sharp line separating the halves of the field of vision is brought into focus. This being accomplished an observation tube filled with pure water is placed in the apparatus and the telescope again adjusted to bring the dividing line of the field into focus. The beginner especially, should repeatedly study this adjustment and be impressed with the fact that only in a sharply defined field are practical observations of any worth. The importance of having all the lenses perfect and all the cover glasses without a flaw may be fully appreciated when it is remembered that the polarized ray, already deprived of half its original luminous power, must pass through several centimeters of crystallized calcium carbonate, and half a dozen disks of glass and quartz, and as many lenses before reaching the eye of the observer. Only with the greatest care and neatness is it possible to secure the required degree of illumination. The zero point having been well studied and accurately adjusted, the scale of the instrument may be tried with a series of quartz plates of known polarizing power at the temperature of the observation. In the apparatus with double quartz wedge compensation, it will be noticed that the marks on one scale are black and on the other red. The black is the working and the red the control scale. To operate this instrument, the red scale is placed exactly at the zero point. The black scale is also placed at zero, and if the field of vision is not neutral, it is made so by the micrometer screw with which the black scale is provided. In a right-handed solution, the red scale is left at zero and the black one moved to the right until neutrality in the field of vision is reached and the reading is taken. The observation tube containing the sugar solution is taken out and the red scale moved until the field of vision is again neutral and the reading of the red scale taken. The two readings should agree. Any failure in the agreement shows some fault either in adjusting the apparatus or in its construction, or some error in manipulation.
The double compensating shadow instruments are more readily tested for accuracy in all parts of the scale than those of any other construction. The two compensating wedges are cut with the greatest care, one from a left-handed and the other from a right-handed perfectly homogeneous quartz crystal. Since faults in these wedges are due either to lack of parallelism of surface, or of perpendicularity to the optical axis of the crystal, and since these faults of crystallization or construction must be in a very limited degree common they would not coincide once in many thousand times in the two wedges. This is easily shown by the theory of probabilities. If, therefore, the two readings made at any point, should not agree, it must be due either to a fault in one of the wedges, or to a fault in reading or a lack of adjustment, as has been mentioned. In such cases the readings should be retaken and the errors are usually easily discovered.
78. Control Observation Tube.—Instead of using quartz plates of known values for testing the accuracy of the scale, an observation tube may be used, the length of which can be varied at the pleasure of the observer.
The construction of a tube of this kind is shown in [Fig. 40]. The tube B is movable telescopically in A by means of the ratchet wheel shown. It is closed at D water-tight by a glass disk. The tube B fits as accurately into A as is possible to permit of free movement, and any liquid which may infilter between its outer surface and the inner surface of A is prevented from gaining exit by the washer C, which fits both tubes water-tight. The ratchet which moves B in A carries a millimeter scale and vernier N whereby the exact thickness of the liquid solution between the surfaces of the glass disks D and E can be always determined.
Figure 41. Control Observation Tube.
By this device the length of liquid under observation can be accurately read to a tenth of a millimeter. The cover glass E is held in position by any one of the devices in common use for this purpose in the case in question, by a bayonet fastening. The funnel T, communicating directly with the interior of A, serves to hold the solution, there being always enough of it to fill the tube when D is removed to the maximum distance from C, which is usually a little more than 200 millimeters.
Let the control tube be adjusted to 200 millimeters and filled with a solution of pure sugar, which reads 100 per cent or degrees in a 200 millimeter tube. Since the degree of rotation is, other things being equal, proportional to the length of the column of polarizing solution, it follows that if the tube B be moved inward until the distance between D and C is 100 millimeters, the scale should read 50° or per cent. By adjusting the length of the distance between B and C it is easily seen that every part of the scale can be accurately tested.
The tube should be filled by removing the funnel and closing the orifice with a screw cap which comes with the apparatus. The cap E is then removed and the tube filled in the ordinary manner. This precaution is practiced to avoid carrying air bubbles into the tube when filled directly through the funnel. With a little care, however, this danger may be avoided, or should air bubbles enter they can be easily removed by inclining the tube.
In case the solution used be not strictly pure it may still be employed for testing the scale. Suppose, for instance, that a solution made up in the usual way, has been made from a sample containing only 99.4 per cent of sugar. Then in order to have this solution read 100° on the scale the tube should be set at 201.2 millimeters, according to the formula
| 200 × 100 | = 201.2. | |
| 99.4 |
By a similar calculation the position of the tube for reading any desired degree on the scale can be determined. The importance of controlling all parts of the scale in compensating instruments is emphasized by the fact that a variation of only 0.016 millimeter in the thickness of the compensating wedge will cause a change of one degree in the reading of the instrument.
79. Setting the Polariscope with Quartz Plates.—Pure sugar is not always at the command of the analyst, and it is more convenient practically to adjust the instrument by means of quartz plates, the sugar values of which have been previously tested for the character of the light used. Assuming the homogeneity of a plate of quartz, the degree of deflection which it imparts to a plane of polarized light depends on the quality of the light, the thickness of the plate, and the temperature.
In respect of the quality of light, red polarized rays are least, and violet most deflected. The degree of rotation produced with any ray, at a given temperature, is directly proportional to the thickness of the plate. Temperature affects the rotating power of a quartz plate in a degree highly significant from a scientific point of view and not wholly negligible for practical purposes. The rotating power of a quartz plate increases with the temperature and the variation may be determined by the formula given below:[44]
The formula is applicable for temperatures between 0° and 100°. Its values are expressed in degrees of angular measure which can be converted into degrees of the sugar scale by appropriate factors:
Formula.— aᵗ = a°(1 + 0.000146t);
in which a° = polarization in angular degrees at 0°, t the temperature of observation and aᵗ the rotation desired.
Example.—A quartz plate which has an angular rotation of 33° at 0° will have a rotation at 20° of 33°.09834.
aᵗ = 33(1 + 0.000146 × 20) = 33.09834.
Since in instruments using the ventzke scale one degree of the sugar scale is equal to 0.3467 degree angular measure, the sugar value of the quartz plate mentioned is equal to 95.47 percent; 33.09834 ÷ 0.3467 = 95.47.
The sugar value of this plate at 0° is 95.18 per cent; 33 ÷ 0.3467 = 95.18.
80. Tables for Correcting Quartz Plates.—Instead of calculating the variation in quartz plates for each temperature of observation, it is recommended by the Bureau of Internal Revenue of the Treasury, to use control quartz plates the values of which at any given temperature, are found on a card which accompanies each one.[45] The variations given, are from temperatures between 10° and 35°. Three control plates are provided with each instrument used by the Bureau, for polarimetric work in the custom houses, or in ascertaining bounties to be paid on the production of domestic sugars. For example, the case of a sugar which polarizes 80°.5 may be cited. One of the control plates nearest to this number, is found to have at the temperature of observation, a polarization of 91°.4, the reading being made in each case at 25°. On consulting the card which accompanies the control plate, it is seen that its value at the temperature mentioned, is 91°.7. The reading of the instrument is therefore too low by three-tenths of a degree, and this quantity should be added to the observed polarization, making it 80°.8. In this method of correcting the reading for temperature, it is assumed that the compensating wedges of the instrument, are free of error at the points of observation. The plates used for the purpose above, are all standardized in the office of weights and measures of the Coast and Geodetic Survey, before delivery to the analysts.
81. Applicability of Quartz Plates.—Quartz plates which are correctly set for one instrument or kind of light, should be equally accurate for the sugar scales of all instruments, using the same sugar factor. In other words a quartz plate which reads 99° on a scheibler color polariscope, should give the same reading on the sugar scale of a shadow compensating or a monochromatic direct reading apparatus using 26.048 grams of sugar.
The most useful quartz plates for sugar analysis, are those which give the readings at points between 80° and 96°, which cover the limits of ordinary commercial sugars. For molasses the plates should read from 45° to 55°. For sugar juices of the cane and beet, the most convenient graduation would be from 10° to 20°, but plates of this value would be too thin for practical work and are not in use. When quartz plates are to be used for control purposes, they should be purchased from reliable manufacturers, or better, tested directly against pure sugar solutions by the observer.
In practice we have found quartz plates as a rule, true to their markings.
82. The Sugar Flask.—Sugar solutions are prepared for polarization in flasks graduated to hold fifty or one hundred cubic centimeters. For scientific work a flask is marked to hold 100 grams of distilled water at 4°. The weights are all to be reduced to a vacuum standard. One flask having been marked in this way, others may be compared directly therewith by means of pure mercury. For this purpose the flasks must be perfectly dry and the mercury pure, leaving no stain on the sides of the flask. The glass must also be strong enough to undergo no change in shape from the weight of mercury used.
For sugar work the true 100 gram flask is not usually employed, but one graduated by weighing at 17°.5. These flasks are graduated by first weighing them perfectly dry, filling with distilled water and again weighing fifty and fifty-five, or 100 and 110 grams of water at the temperature named. Since the volume of water at 17°.5 is greater than at 4° the sugar flask in ordinary use has a greater volume by about 0.25 cubic centimeter than the true flask. The observer should always secure a statement from the dealer in respect of the volume of the flask used in testing the scale of the polariscope purchased. In the graduation of a flask in true cubic centimeters, when brass weights are used it will be necessary to correct the weight of each gram of water by adding to it one milligram, which is almost exactly the weight of the volume of air displaced by one gram of water in the circumstances named. If the flask be first counterbalanced and it be desired to mark it at 100 cubic centimeters the sum of the weights placed in the opposite pan should be 100 - 0.100 = 99.900 grams. While this is not a rigidly exact correction it will be sufficient for all practical purposes. A liter of dry air weighs 1.29366 grams; and 100 cubic centimeters of water would therefore displace 0.129 gram of air. But the brass weights also displace a volume of air which when deducted reduces the correction to be made for the water to nearly the one named. For convenience in inverting sugar solutions the flasks used in practical work are graduated at fifty and fifty-five and 100 and 110 cubic centimeters respectively.
83. Preparing Sugar Solutions for Polarization.—If sugar samples were always pure the percentage of sugar in a given solution could be directly determined by immediate polarization. Such cases, however, are rarely met in practice. In the majority of cases the sample is not only to be brought into solution but is also to be decolorized and rendered limpid by some one of the methods to be described. A perfectly limpid liquid is of the highest importance to secure correct observations. With a cloudy solution the field of vision is obscured, the dividing line of the two halves, or the double line in the triple field, becomes blurred or invisible and the intensity of illumination is diminished. A colored liquid which is bright is far more easy to polarize than a colorless liquid which is turbid. In fact, it is only rarely in sugar work that samples will be found which require any special decolorizing treatment other than that which is received in applying the reagents which serve to make the solutions limpid. In the following paragraphs the approved methods of clarifying sugar solutions preparatory to observation in the polariscope will be described.
84. Alumina Cream.—The hydrate of alumina, commonly known as alumina cream, is always to be preferred as a clarifying agent in all cases where it can be successfully applied.[46] It is a substance that acts wholly in a mechanical way and therefore leaves the sugars in solution unchanged, carrying out only suspended matters. In the preparation of this reagent a solution of alum is treated with ammonia in slight excess, the aluminum hydroxid produced washed on a filter or by decantation until neutral in reaction. The hydroxid is suspended in pure water in proportions to produce a creamy liquid. Although apparently very bulky, the actual space occupied by the amount of dry hydroxid added in a few cubic centimeters is so small as to produce no disturbing effect of importance on the volume of the sugar solution. The cream thus prepared is shaken just before using and from one to five cubic centimeters of it, according to the degree of turbidity of the saccharine solution, are added before the volume in the flask is completed to the mark. After filling the flask to the mark the ball of the thumb is placed over the mouth and the contents well shaken and allowed to stand for a few moments before filtering.
The alumina cream is well suited to use with solutions of commercial sugars of not too low a grade and of most honeys and high grade sirups. It is usually not powerful enough to clarify beet and cane juices, molasses and massecuites.
85. Basic Lead Acetate.—A solution of basic lead acetate is an invaluable aid to the sugar analyst in the preparation of samples for polarimetric observation. It acts as a clarifying agent by throwing out of solution certain organic compounds and by uniting with the organic acids in solution forms an additional quantity of precipitate, and these precipitates act also mechanically in removing suspended matters from solution. The action of this reagent is therefore much more vigorous than that of alumina cream. Coloring matters are often precipitated and removed by treatment with lead acetate. It happens therefore that there are few samples of saccharine bodies whose solutions cannot be sufficiently clarified by lead acetate to permit of polarimetric observation.
The reagent is most frequently employed of the following strength:[47] Boil for half an hour in one and a half liters of water 464 grams of lead acetate and 264 grams of litharge with frequent stirring. When cool, dilute with water to two liters, allow to stand until clear, and decant the solution. The specific gravity of this solution is about 1.267.
In a solution of basic lead acetate of unknown strength the percentage of lead acetate may be determined from its specific gravity by the following table:[48]
Percentage of Lead Acetate Corresponding
to Different Specific Gravities at 15°.
| Specific gravity. | Percentage of lead acetate. | Specific gravity. | Percentage of lead acetate. |
|---|---|---|---|
| 1.0127 | 2 | 1.2040 | 28 |
| 1.0255 | 4 | 1.2211 | 30 |
| 1.0386 | 6 | 1.2395 | 32 |
| 1.0520 | 8 | 1.2579 | 34 |
| 1.0654 | 10 | 1.2768 | 36 |
| 1.0796 | 12 | 1.2966 | 38 |
| 1.0939 | 14 | 1.3163 | 40 |
| 1.1084 | 16 | 1.3376 | 42 |
| 1.1234 | 18 | 1.3588 | 44 |
| 1.1384 | 20 | 1.3810 | 46 |
| 1.1544 | 22 | 1.4041 | 48 |
| 1.1704 | 24 | 1.4271 | 50 |
| 1.1869 | 26 |
86. Errors Due to use of Lead Solutions.—In the use of lead solutions there is danger of errors intruding into the results of the work. These errors are due to various sources. Lead subacetate solution, when used with low grade products, or sugar juices, or sirups from beets and canes, precipitates albuminous matters and also the organic acids present. The bulk occupied by these combined precipitates is often of considerable magnitude, so that on completing the volume in the flask the actual sugar solution present is less than indicated. The resulting condensation tends to give too high a polarimetric reading. With purer samples this error is of no consequence, but especially with low grade sirups and molasses it is a disturbing factor, which must be considered.
One of the best methods of correcting it has been proposed by Scheibler.[49] To 100 cubic centimeters of a solution of the sample, ten of lead solution are added, and after shaking and filtering the polarimetric reading is taken. Another quantity of 100 cubic centimeters of the solution with ten of lead is diluted to 220 cubic centimeters, shaken, filtered, and polarized. Double the second reading, subtract it from the first, multiply the difference by 2.2, and deduct the product from the first reading. The remainder is the correct polarization.
The process just described is for the usual work with beet juices and sirups. For cane juices measured by the graduated pipette, hereafter to be described, and for weighed samples of molasses and massecuites, the following method of calculation is pursued.[50] To the sample dissolved in water, add a measured portion of the lead subacetate solution, make its volume 100 cubic centimeters and observe the polarimetric reading. Prepare a second solution in the same way and make the volume double that of the first and again take the polarimetric reading. Multiply the second reading by two, subtract the product from the first reading and multiply the remainder by two, and subtract the product from the first reading.
| Example.— | First polarization | 30.0 | |
| Second polarization | 14.9 | ||
| Then 30 - (2 × 14.9 = 29.8) | = 0.2 | ||
| 0.2 × 2 | = 0.4 | ||
| and 30 - 0.4 | = 29.6 |
The corrected reading therefore shows that the sample contained 29.6 per cent of sugar.
87. Error Due to Action of Lead Subacetate on Levulose.—In the use of lead subacetate solution not only is there danger of error due to the causes just described, but also to a more serious one, arising from the chemical interaction of the clarifying agent and levulose.[51]
Lead subacetate forms a chemical union with levulose and the resulting compound has a different rotatory power from the left-handed sugar in an uncombined state. By adding a sufficient quantity of subacetate solution, the left-handed rotation of levulose may be greatly diminished if not entirely destroyed. In this case the dextrose, which with levulose forms inverted sugar, serves to increase the apparent right rotation due to the sucrose in solution. The reading of the scale is therefore higher than would be given by the sucrose alone. If the lead subacetate could be added in just the proportion to make the invert sugar neutral to polarized light, its use would render the analysis more accurate; but such a case could only arise accidentally. To correct the error, after clarification, the compound of levulose and lead may be decomposed by the addition of acetic acid according to the method of Spencer. In this case the true content of sucrose can only be obtained by the method of inversion proposed by Clerget, which will be described in another paragraph.
88. Clarification with Mercuric Compounds.—Where the disturbing bodies in a solution are chiefly of an albuminoid nature, one of the best methods of securing clarification is by the use of a solution containing an acid mercuric compound.[52] In the case of milk this method is to be preferred to all others. Albuminoid bodies themselves, have the property of deflecting the plane of polarization, as a rule, to the left, and therefore, should be completely removed from solutions containing right-handed sugars such as lactose. For this purpose the mercuric compound is more efficient than any other. It is prepared and used as follows.[53] Dissolve mercury in double its weight of strong nitric acid and dilute the solution with an equal volume of water. One cubic centimeter of this solution is sufficient to clarify fifty times its volume of milk.
89. Decolorization by Means of Bone-Black.—Where the means already described fail to make a solution sufficiently colorless to permit of the passage of a ray of polarized light, recourse should be had to a decolorizing agent. The most efficient of these is bone-black. For laboratory work it is finely ground and should be dry if added to an already measured solution. When moist it should be added to the flask before the volume is completed, and a correction made for the volume of the dry char employed. Bone-black has the power of absorbing a certain quantity of sugar, and for this reason as little of it should be employed as is sufficient to secure the end in view. If not more than one gram of the char be used for 100 cubic centimeters of solution, the error is not important commercially. The error may be avoided by placing the char on the filter and rejecting the first half of the filtered solution. The char becomes saturated with the first portion of the solution, and does not absorb any sugar from the second. This method, however, does not secure so complete a decolorization as is effected by adding the black directly to the solution and allowing to stand for some time with frequent shaking.
90. Remarks on Analytical Process.—Since large weights of sugar are taken for polarization, a balance which will weigh accurately to one milligram may be used. In commercial work the weighing is made in a counterpoised dish with a prominent lip, by means of which the sample can be directed into the mouth of the flask after partial solution. Where the air in the working room is still, an uncovered balance is most convenient. With a little practice the analyst will be able to dissolve and transfer the sample from the dish to the flask without danger of loss. The source of light used in polarizing should be in another room, and admitted by a circular opening in the partition. In a close polarizing room, which results from the darkening of the windows, the temperature will rapidly rise if a lamp be present, endangering notably the accuracy of the work, and also interfering with the comfort of the observer. The greatest neatness must be practiced in all stages of the work, and especially the trough of the polariscope must be kept from injury which may arise from the leaking of the observation tubes. Dust and dirt of all kinds must be carefully excluded from the lenses, prisms, wedges and plates of the instrument.
91. Determination of Sucrose by Inversion.—In the foregoing paragraphs directions have been given for the estimation of sugar (sucrose) by its optical properties. It has been assumed so far, that no other disturbing bodies have been present, save those which could be removed by the clarifying agents described. The case is different when two or more sugars are present, each of which has a specific relation to polarized light. In such cases some method must be used for the optical determination of sucrose, which is independent of the influence of the other polarizing bodies, or else recourse must be had to other methods of analysis. The conversion of the sucrose present into invert sugar by the action of an acid or a ferment, affords an opportunity for the estimation of sucrose in mixed sugars, by purely optical methods. This process rests upon the principle that by the action of a dilute acid for a short time, or of a ferment for a long time, the sucrose is completely changed, while other sugars present are not sensibly affected. Neither of these assumptions is rigidly correct but each is practically applicable.
The sucrose by this process of hydrolysis is converted into an equal mixture of levulose and dextrose. The former, at room temperatures, has the higher specific rotating power, and the deflection of the plane of polarization in a solution of inverted sugar is therefore to the left. The levorotatory power of invert sugar varies with the temperature, and this arises from the optical properties of the levulose. The influence of temperature on the rotating power of other sugars, is not imperceptible in all cases, but in practice is negligible.
This method of analysis is invaluable in control work in factories, in the customs and in agricultural laboratories. Since the rotating power of levulose diminishes as the temperature rises, an accurate thermometric observation must accompany each polarimetric reading. At about 88° the rotatory powers of dextrose and levulose are equal, and a solution of pure invert sugar examined at that degree, is found to be neutral to polarized light.
92. Clerget’s Method of Inversion.—The classical method of Clerget for the determination of cane sugar by double polarization before and after inversion, was first described in a memoir presented to the Society of Encouragement for National Industry on the 14th of October, 1846. The following description of the original method is taken from a reprint of the proceedings of that Society, dated Nov. 1846:
Clerget points out first the observation of Mitscherlich regarding the influence of temperature on the rotatory power of invert sugar, and calls attention to the detailed experiments he has made which resulted in the determination of the laws of the variation. From these studies he was able to construct a table of corrections, applicable in the analysis of all saccharine substances in which the cane sugar is polarized before and after inversion. The basis of the law rests upon the observation that a solution of pure sugar, polarizing 100° on the sugar scale, before inversion, will polarize 44° to the left after inversion at a temperature of zero. The quantity of sugar operated upon by Clerget amounted to 16.471 grams in 100 cubic centimeters of liquid. On the instrument employed by him this quantity of sugar in 100 cubic centimeters gave a reading of 100° to the right on the sugar scale when contained in a tube twenty centimeters in length. The process of inversion carried on by Clerget is as follows:
The sugar solution is placed in a flask, marked on the neck at 100 and 110 cubic centimeters; or if smaller quantities are used, in a flask marked on the neck at fifty and fifty-five cubic centimeters. The flask is filled with the sugar solution to the first mark and then a sufficient quantity of strong hydrochloric acid added to bring the volume of the liquid to the second mark. The mouth of the flask is then closed with the thumb and its contents thoroughly mixed by shaking. A thermometer is placed in the flask which is set in a water-bath in such a way that the water comes just above the level of the liquid in the neck of the flask. The water is heated in such a manner as to bring the temperature of the contents of the flask, as determined by the thermometer, exactly to 68° and at such a rate as to require fifteen minutes to reach this result. At the end of fifteen minutes the temperature having reached 68° the flask is removed and placed at once in another water-bath at the temperature of the room, to which temperature the contents of the flask are cooled as rapidly as possible. To make the polarimetric observation a tube twenty-two centimeters in length is filled with the inverted sugar solution by means of a tubulure in its center, which serves not only the purpose of filling the tube but also afterwards to carry the thermometer, by means of which the temperature of observation can be taken. If the sugar solution be turbid, or contain any lead chlorid due to the previous use of basic lead acetate in clarification, it should be filtered before being introduced into the observation tube. This tube being one-tenth longer than the original compensates for the dilution caused by the addition of the hydrochloric acid in inversion.
When reading, the bulb of the thermometer should be withdrawn far enough to permit the free passage of the ray of light and the exact temperature of the solution noted.
The above outline of Clerget’s method of inversion is given in order that the analyst may compare it with any of the variations which he may find in other works. The chief points to which attention is called, are, first, the fact that only a little over sixteen grams of sugar are used for ten cubic centimeters of strong hydrochloric acid, and second, that the time of heating is exactly fifteen minutes, during which time the contents of the flask should be raised from room temperature to exactly 68°.
From the above it is seen that the process of Clerget, as originally described, can be applied directly to all instruments, using approximately sixteen grams of sugar in 100 cubic centimeters. Experience has also shown that even when larger quantities of sugar are employed, as for instance, approximately twenty-six grams, the inversion is effected with practical completeness in the same circumstances. It is advised, therefore, that in all analytical processes, in which cane sugar is to be determined by the process of inversion with an acid, the original directions of Clerget be followed as strictly as possible. Experience has shown that no one of the variations proposed for Clerget’s original method has any practical advantage and the analyst is especially cautioned against those methods of inversion in which the temperature is continued at 68° for fifteen minutes or in which it is allowed to go above that degree.
93. Influence of Strength of Solution and Time of Heating on the Inversion of Sucrose.—As has been intimated, the strength of a sugar solution and the time of heating with hydrochloric acid are factors that must be considered in determining a formula for the calculation of sucrose by inversion. The Clerget formula holds good only for the conditions specified and these conditions must be rigidly adhered to in order to secure the proper results. This matter has been thoroughly studied by Bornträger, who also gives a nearly complete bibliography of the subject.[54] As a result of his investigations it seems well established that the original Clerget formula is practically correct for the conditions indicated, Bornträger modifying it only by substituting in the formula 143.66 for 144. This is so nearly the same as the Clerget factor that it is not advisable to substitute it therefor. If, however, the inverted sugar solution be diluted to double its volume before polarization the factor proposed by Landolt, viz., 142.4, gives more nearly accurate results. If the hydrochloric acid be neutralized before polarization by an alkaline body, the character of the salt which is formed also influences, to a greater or less extent, the specific rotatory power of the solution. Hydrochloric acid itself also influences the rotation to a certain degree.[55]
94. Calculation of Results.—The percentage of sucrose in a solution which has been polarized before and after inversion is calculated by an appropriate formula from the data obtained or is taken directly from tables. These tables are too long to insert here, and in point of fact the calculation can be made from the formula almost as quickly as the result can be taken from a table.
Two factors are commonly used in the calculations, one based on the supposition that a sugar solution polarizing 100° to the right will, after inversion, give a reading of 44° to the left, at zero temperature. In the second formula in common use the polarization to the left in the circumstances mentioned above is assumed to be 42.4, a number reached by Landolt after a long series of experiments.[56] The principle of the calculation of the percentage of sucrose is based upon the original observation of Clerget to the effect that the algebraic difference of the two readings, divided by 144, less half of the temperature, will give the percentage of sucrose desired. The formula by which this is obtained is
| S = | a - b | . | |
| K - | t | ||
| 2 | |||
In this formula a is the polarization on the sugar scale before inversion, b the polarization after inversion, K the constant representing the algebraic difference of the two polarizations of pure sugar at 0° and t the temperature of the observation. To K may be assigned the values 144 or 142.4, the one in more common use. In case the polarization, after inversion, is to the left, which is more commonly the case, the sum of the two readings is taken for a - (-b) = a + b; when both polarizations are to the right or left the difference is taken. S is the percentage of sucrose desired.
| Example.— | Let the polarization before inversion be | +95 |
| and after inversion | -26 | |
| and the temperature | 20° |
| Then S = | 95 + 26 | = 121 ÷ 134 = 90.6. |
| 144 - 10 |
Substituting the value 142.4 for K, the result of the calculation is 91.4.
In high grade sugars, therefore, the difference in the results secured by taking the two values of K amounts to about 1 per cent of sucrose.
For a further discussion of the theory and practice of inversion the reader is referred to the articles of Herles, Herzfeld, and Wohl.[57]
95. Method Of Lindet.—Courtonne recommends the method of Lindet for securing the inversion instead of the method of Clerget.[58] Modified by Courtonne, the method is as follows:
Make two or three times the normal weight of sugar dissolved in water to a volume of 200 or 300 cubic centimeters, as the case may be. After thoroughly mixing proceed as follows:
First, to Obtain the Polarization Direct.—Place fifty or 100 cubic centimeters of the prepared solution in a flask marked at fifty and fifty-five or at 100 and 110 cubic centimeters, add a sufficient quantity of lead acetate to secure a complete clarification, make the volume to fifty-five or 110 cubic centimeters, shake thoroughly, filter, and polarize in a 220 millimeter tube.
Second, to Obtain the Rotation after Inversion.—Place twenty cubic centimeters of the original solution, in a flask marked at fifty cubic centimeters, containing five grams of powdered zinc. The flask should be placed in boiling water. Add, little by little so as to avoid a too rapid evolution of hydrogen, ten cubic centimeters of hydrochloric acid made of equal parts of the strongest acid and water. After the operation is terminated, cool to the temperature of the room, make the volume to fifty cubic centimeters, polarize, and determine the rotation. The volume occupied by the zinc which is not dissolved, will be about one-half cubic centimeter, hence the deviation should be multiplied by the factor 2.475 in order to get the true deviation which would have been produced by the pure liquor. We have then:
- A = the deviation direct.
- B = the deviation after inversion.
- C = the algebraic difference of the deviations.
The amount of sucrose, therefore, would be calculated by the formula of Creydt,[59]
| X = | C - 0.493A | ; |
| 0.827 |
for raffinose the formula would be
| Y = | A - S | ; |
| 1.57 |
in which S is the deviation due to the sucrose present. The solutions inverted in the manner described are absolutely colorless. There is no need of employing bone-black to secure the saccharimetric reading nor does it present any uncertainty. It is thought by Courtonne that this method will soon take the place of the method of Clerget on account of the advantages above mentioned. The method will be somewhat improved by adopting the following suggestions:
1. Instead of allowing any arbitrary number for the volume of the undissolved zinc, decant the liquid, after inversion, into another flask and wash repeatedly with hot water until all trace of sugar is removed from the flask in which the inversion took place.
2. Instead of polarizing in a 200 millimeter tube make the observation in a 500 millimeter tube, which will permit of the reading being made without any correction whatever.
96. Inversion by Means of Invertase.—Instead of using acids for the inversion of cane sugar the hydrolysis can be easily effected by means of a ferment derived from yeast. A complete history of the literature and characteristics of this ferment, together with a study of its properties and the various methods of preparing it, has been given by O’Sullivan and Tompson.[60] In the preparation of invertase, the method found most effective is the following:
The yeast is allowed to liquify for at least a month in a fairly warm room without stirring. At the end of this time the surface is removed and any supernatant liquid poured away. The lower sedimentary part is thrown on a quick-acting filter and allowed to drain for two days. To the filtrate, alcohol of specific gravity 0.87 is gradually added to the extent of one and a half times its volume, with continued and vigorous stirring. The process of adding the alcohol and stirring should require about half an hour, after which the mixture is allowed to stand for twenty-four hours to allow the precipitated invertase to settle. The supernatant liquid is poured away and the precipitate washed several times on successive days by decantation with alcohol of 0.92 specific gravity. When the washings become nearly colorless the precipitate is thrown on a filter, allowed to drain, and immediately removed and mixed with a large bulk of alcohol of 0.92 specific gravity. The precipitate is again collected, mixed thoroughly with its own bulk of water, and some alcohol of 0.97 specific gravity, allowed to stand for a few hours and thrown on a filter. The filtrate contains the invertase.
97. Determination of Activity of Invertase.—The activity of a solution of invertase, prepared as above, is measured by the number of minutes required for it to reduce to zero the optical power of a solution of 100 times its weight of cane sugar at a temperature of 15°.5. In order to facilitate the action of the invertase, a trace of sulfuric acid is added to the solution. The manipulation is as follows:
Fifty grams of sucrose are dissolved in water and made to a volume of nearly a quarter of a liter and placed in a bath maintained at 15°.5. Half a gram of the invertase is added, the time noted, the solution immediately made up to a quarter of a liter and well shaken. The contents of the flask are poured rapidly into five beakers; the actual quantity in each beaker is not necessarily the same. To each of these beakers, in succession, are added the following amounts of decinormal sulfuric acid, viz., one-tenth, three-tenths, six-tenths, one, and one and four-tenths cubic centimeters. After an hour a small quantity of the solution is taken from beaker No. 3 and the reaction of the invertase stopped by adding a few drops of strong potassium hydroxid and the time of adding this reagent noted. This solution is then read in the polariscope and the percentage of sugar inverted is calculated from the formula C₁₂H₂₂O₁₁ + H₂O = C₆H₁₂O₆ + C₆H₁₂O₆.
The calculation of the amount of cane sugar inverted is based on the formula,
(38.4 - d) ÷ 0.518 = p.
In this formula d equals the divisions of the sugar scale read on the polariscope; p the percentage of cane sugar inverted; 38.4 the reading on the sugar scale of the original sugar solution and 51.8 the total number of divisions of the cane sugar scale that the polariscope reading would fall through if all the sugar were inverted. The observation tubes used in the polarization are only 100 millimeters in length. After stopping the action of the invertase with potassium hydroxid the solution is allowed to stand for some time before polarization inasmuch as the dextrose formed appears to assume the state of birotation and some time is required for it to reach its normal rotatory power. If the invertase be used in the alcoholic solution a sufficient quantity should be added to be equivalent to 0.01 of the sucrose present. The time which the contents of beaker No. 3 will take to reach optical activity is calculated in a manner described by O’Sullivan and Tompson, but too long to be inserted here.[61] The five beakers mentioned above are examined in succession and the amount of sulfuric acid best suited to the maximum inversion thus determined. This quantity is then used in subsequent hydrolyses with the given sample of invertase.
The action of invertase on sucrose is very rapid at the first and becomes very much slower towards the end. At a temperature of 15°.5 it is advisable to let the solution stand for forty-eight hours in order to be sure that complete inversion has taken place. For this reason the method by inversion by means of invertase is one of no great practical importance, but it may often be useful to the analyst when the employment of an acid is inadmissible.
98. Inversion by Yeast.—Owing to the difficulty of preparing invertase, O’Sullivan and Thompson[62] propose to use yeast as the hydrolytic agent, as first suggested by Kjeldahl. It is shown that in the use of yeast it is not necessary to employ thymol or any other antiseptic. The method of procedure is as follows: The cane sugar solution of usual strength should not be alkaline, but, if possible, should be exactly neutral. If there be any ferment suspected, the temperature should be momentarily raised to 80° to destroy its activity. The polariscopic reading of the solution is then taken at 15°.5 and the amount of copper reduced by the solution should also be determined.
Fifty cubic centimeters of the solution are poured into a beaker and raised to a temperature of 55° in a constant temperature bath. Some brewers yeast amounting to about one-tenth of the total amount of sugar to be inverted, pressed in a towel, is thrown into the hot solution and the whole stirred until mixture is complete. The solution is left for four hours in the water-bath, at the end of this time it is cooled to 15°.5, a little freshly precipitated aluminum hydroxid added, and the volume made to 100 cubic centimeters. A portion of this solution is filtered and its polariscopic reading observed. The solution is then left till the next day, when another polariscopic reading is taken in order to prove that inversion is complete. The copper reducing power is also determined. The method of calculating the results is the same as when invertase is used. The following formulas are employed.
a = the number of divisions indicated by the polariscopic reading for a 200 millimeter tube:
aʹ = the same number after inversion:
m = the number of the divisions of the polariscopic scale which 200 millimeters of the sugar solution containing one gram of cane sugar per 100, alter at 15°.5 on being inverted: In the case of the ventzke polarimeter scale, one gram of cane sugar in 100 cubic centimeters, indicates +3.84 divisions and after inversion it gives -1.34 div. In experiments of this kind, therefore, m = 5.18.
P = the weight of cane sugar present in 100 cubic centimeters of the original solution:
The formula employed then is
| P = | a - 2aʹ | . |
| m |
For the copper reduction data the following are used:
G = the weight of 100 cubic centimeters of the original solution:
Gʹ; = the same for the inverted solution: Allowance must be made here both for the dilution and for the 5 per cent increase of the inverted sugar, but the latter number is so small that it need not be calculated accurately.
w = the weight of the original solution used for the estimation:
wʹ = the same factor for the inverted solution:
k = the weight of cupric oxid reduced by w:
kʹ = the same factor for wʹ:
p = the weight of cane sugar present in 100 cubic centimeters of the original solution: The formula to be employed then is
p = 0.4308  2 | Gʹ kʹ | - | G k |  . |
| wʹ | w |
This method has been applied to the estimation of cane sugar in molasses, apple juices and other substances. It is recommended by the authors as a simple and accurate means of estimating sucrose in all solutions containing it. The methods of making the copper reductions will be given hereafter.
99. Application of the Process.—In practice the process of inversion is used chiefly in the analysis of molasses and low grade massecuites. In approximately pure sugars the direct polarization is sufficiently accurate for all practical purposes. In molasses resulting from the manufacture of beet sugar are often found considerable quantities of raffinose, and the inversion process has been adapted to that character of samples. In molasses, in sugar cane factories, the disturbing factors are chiefly invert sugars and gums. The processes used for molasses will be given in another paragraph. In certain determinations of lactose the process of inversion is also practiced, but in this case the lactose is converted into dextrose and galactose, and the factors of calculation are altogether different. The process has also been adapted by McElroy and Bigelow to the determination of sucrose in presence of lactose, and this method will be described further on. In general the process of inversion is applicable to the determination of sucrose in all mixtures of other optically active bodies, which are not affected by the methods of inversion employed.
100. Determination of Sucrose and Raffinose.—Raffinose is a sugar which often occurs in beets, and is found chiefly in the molasses after the chief part of the sucrose has been removed by crystallization. It is also found in many seeds, notably in those of the cotton plant. In a pure solution of sucrose and raffinose, both sugars may be determined by the inversion method of Creydt.[63] The inversion is effected by means of hydrochloric acid in the manner described by Clerget. The following formulas are calculated for a temperature of observation of 20°, and the readings should be made as near that temperature as possible.
| (1)S = | C - 0.493A |
| 0.827 |
| (2) R = | A - S | = 1.017A - | 6 |
| 1.57 | 1.298 |
In these formulas S and R are the respective per cents of sucrose and raffinose desired, A the polarization in sugar degrees before inversion, B the polarization after inversion read at 20°, and C is the algebraic difference between A and B. It must be understood that these formulas are applicable only to a solution containing no other optically active substances, save sucrose and raffinose.
101. Specific Rotatory Power.—In order to compare among themselves the rotations produced on a plane of polarized light by different optically active bodies in solution, it is convenient to refer them all to an assumed standard. The degree of rotation which the body would show in this condition, is found by calculation, since, in reality, the conditions assumed are never found in practice. In the case of sugars and other optically active bodies, the standard of comparison is called the specific rotatory power. This factor in any given case, is the angular rotation which would be produced by any given substance in a pure anhydrous state if it were one decimeter in length and of a specific gravity equal to water. These are conditions which evidently do not exist in the case of sugars, since crystalline sugar particles have no polarizing power, and it would be impossible to pass a ray of light through an amorphous sugar column of the length specified. The specific rotatory power is therefore to be regarded as a purely theoretical factor, calculated from the actual data obtained by the examination of the solution of any given substance. If the length of the observation tube in decimeters be represented by l, the percentage of the polarizing body in 100 grams by p, and the specific gravity of the solution by d, and the observed angle of rotation by a, then the factor is calculated from the formula:
| [a]Dj = | a. 100 | . |
| p. d. l. |
The symbols Dj refer to the character of light employed, D indicating the monochromatic sodium flame, and j the transition tint from white light.
If the weight of the polarizing body c be given or known for 100 cubic centimeters of the solution the formula becomes
| [a]Dj = | a. 100 | . |
| c. l. |
The latter formula is the one easier of application since it is only necessary in applying it to dissolve a given weight of the active body in an appropriate solvent and to complete the volume of the solution exactly to 100 cubic centimeters. It is therefore unnecessary in this case to determine the specific gravity.
102. Formulas for Calculating Specific Rotatory Power.—In order to determine the specific rotatory power (gyrodynat[64]) of a given substance it is necessary to know the specific gravity and percentage composition or concentration of its solution, and to examine it with monochromatic polarized light in an instrument by which the angular rotation can be measured. The gyrodynat of any body changes with its degree of concentration, in some cases with the temperature, and always with the color of the light. With the red rays the gyrodynat is least and itprogressively increases as the violet end of the spectrum is approached. In practice the yellow ray of the spectrum has been found most convenient for use, and in the case of sugars the gyrodynat is always expressed either in terms of this ray or if made with color compensating instruments in terms of the sensitive or transition tint. In the one case the symbol used is (a)D and in the other (a)j. From this statement it follows that (a)D is always numerically less than (a)j. Unless otherwise specified the gyrodynat of a body is to be considered as determined by yellow monochromatic light, and therefore corresponds to aD.[65]
103. Variations in Specific Rotatory Power.—The gyrodynat of any optically active body varies with the nature of the solvent, the strength of the solution, and the temperature.[66]
Since water is the only solvent of importance in determining the gyrodynat of sugars it will not be necessary here to discuss the influence of the nature of the solvent. In respect of the strength of the solution it has been established that in the case of cane sugar the gyrodynat decreases while with dextrose it increases with the degree of concentration. The influence of temperature on the gyrodynat of common sugars is not of great importance save in the case of levulose, where it is the most important factor, the gyrodynat rapidly increasing as the temperature falls. It is of course understood that the above remarks do not apply to the increase or decrease in the volume of a solution at changed temperatures. This influence of temperature is universally proportional to the change of volume in all cases, and this volumetric change is completely eliminated when the polarizations are made at the temperatures at which the solutions are completed to standard volumes.
104. Gyrodynatic Data for Common Sugars.—In the case of cane sugar the gyrodynat for twenty-five grams of sugar in 100 grams of solution at 20° is [a]D = 66°.37. This is about the degree of concentration of the solutions employed in the shadow lamplight polariscopes. For seventeen grams of sugar in 100 grams of solution the number is [a]D = 66°.49. This is approximately the degree of concentration for the laurent instrument.
For any degree of concentration according to Tollens the gyrodynat may be computed by the following formula: [a]D = 66°.386 + 0.015035p - 0.0003986p², in which p is the number of grams of sugar in 100 grams of the solution.[67] In the table constructed by Schmitt the data obtained are as follows:
| In 100 parts by weight of solution. | Specific gravity | Concentration | Rotation a for 100 mm. | [a]D. | |
|---|---|---|---|---|---|
| Sugar p. | Water q. | at 20° C.d. | c = pd. | at 20° C. | |
| 64.9775 | 35.0225 | 1.31650 | 85.5432 | 56°.134 | 65°.620 |
| 54.9643 | 45.0357 | 1.25732 | 69.1076 | 45°.533 | 65°.919 |
| 39.9777 | 60.0223 | 1.17664 | 47.0392 | 31°.174 | 66°.272 |
| 25.0019 | 74.9981 | 1.10367 | 27.5938 | 18°.335 | 66°.441 |
| 16.9926 | 83.0074 | 1.06777 | 18.1442 | 12°.064 | 66°.488 |
| 9.9997 | 90.0003 | 1.03820 | 10.3817 | 6°.912 | 66°.574 |
| 4.9975 | 95.0025 | 1.01787 | 5.0868 | 3°.388 | 66°.609 |
| 1.9986 | 98.0014 | 1.00607 | 2.0107 | 1°.343 | 66°.802 |
105. Bi-Rotation.—Some sugars in fresh solution show a gyrodynat much higher than the normal, sometimes lower. The former phenomenon is called bi- the latter semi-rotation. Dextrose shows birotation in a marked degree, also maltose and lactose. After standing for a few hours, or immediately on boiling, solutions of these sugars assume their normal state of rotation. The addition of a small quantity of ammonia also causes the birotation to disappear.[68] This phenomenon is doubtless due to a certain molecular taxis, which remains after solution is apparently complete. The groups of molecules thus held in place have a certain rotatory power of their own and this is superadded to that of the normal solution. After a time, under the stress of the action of the solvent, these groups are broken up and the solution then assumes its normal condition.
106. Gyrodynat of Dextrose.—The gyrodynat of dextrose, as has already been mentioned, increases with the degree of concentration, thus showing a property directly opposite that of sucrose.
The general formula for the anhydrous sugar is [a]D = 52.°718 + 0.017087p + 0.0004271p². In this formula p represents the grams of dextrose in 100 grams of the solution. In a ten per cent solution the gyrodynat of dextrose is therefore nearly exactly [a]D20° = 53°. As calculated by Tollens the gyrodynats corresponding to several degrees of concentration are shown in the following table:
| p = grams in 100 grams of solution. | [a]D20° calculated for anhydrous dextrose. | |
|---|---|---|
| 7.6819 | 52°.89 | |
| 9.2994 | 52°.94 | |
| 9.3712 | 52°.94 | |
| 10.0614 | 52°.96 | |
| 10.6279 | 52°.98 | |
| 12.9508 | 53°.05 | |
| 18.6211 | 53°.25 | |
| 31.6139 | 53°.83 | |
| 40.7432 | 54°.34 | |
| 43.9883 | 54°.54 | |
| 53.0231 | 55°.17 | |
| 82.6111 | 57°.80 |
107. Gyrodynats of Other Sugars.—Of the other sugars it will be sufficient to mention only levulose, maltose, lactose, and raffinose. For complete tables of gyrodynatic powers the standard books on carbohydrates may be consulted.[69]
The gyrodynat of levulose is not definitely established. At 14° the number is nearly expressed by [a]D14° = -93°.7.
Invert sugar, which should consist of exactly equal molecules of dextrose and levulose, has a gyrodynat expressed by the formula [a]D0° = -27°.9, with a concentration equivalent to 17.21 grams of sugar in 100 cubic centimeters. The gyrodynat decreases with increase of temperature, according to the formula [a]Dt° = - (27°.9 - 0.32t°). According to this formula the solution is neutral to polarized light at 87°.2, and this corresponds closely to the data of experiment.
Maltose, in a ten per cent solution at 20°, shows a gyrodynat of [a]D20° = 138°.3.
The general formula for other degrees of concentration is [a]D = 140°.375 - 0.01837p - 0.095t, in which p represents the number of grams in 100 grams of the solution and t the temperature of observation.
In the case of lactose [a]D = 52°.53, and this number does not appear to be greatly influenced by the degree of concentration; but is somewhat diminished by a rising temperature.
The gyrodynat of raffinose in a ten per cent solution is [a]D = 104°.5.
CHEMICAL METHODS OF
ESTIMATING SUGARS.
108. General Principles.—The methods for the chemical estimation of sugars in common use depend on the reducing actions exerted on certain metallic salts, whereby the metal itself or some oxid thereof, is obtained. The reaction is either volumetric or the resulting oxid or metal may be weighed. The common method is, therefore, resolved into two distinct processes, and each of these is carried out in several ways. Not all sugars have the faculty of exerting a reducing action on highly oxidized metallic salts and the most common of them all, viz., sucrose is practically without action. This sugar, however, by simple hydrolysis, becomes reducing, but the two components into which it is resolved by hydrolytic action do not reduce metallic salts in the same proportion. Moreover, in all cases the reducing power of a sugar solution is largely dependent on its degree of concentration, and this factor must always be taken into consideration. Salts of copper and mercury are most usually selected to measure the reducing power of a sugar and in point of fact copper salts are almost universally used. Copper sulfate and carbonate are the salts usually employed, and of these the sulfate far more frequently, but after conversion into tartrate. Practically, therefore, the study of the reducing action of sugar as an analytical method will be confined almost exclusively to the determination of its action on copper tartrate.
Direct gravimetric methods are also practiced to a limited extent in the determination of sugars as in the use of the formation of sucrates of the alkaline earths and of the combinations which certain sugars form with phenylhydrazin. Within a few years this last named reaction has assumed a marked degree of importance as an analytical method. The most practical treatment of this section, therefore, for the limited space which can be given it, will be the study of the reducing action of sugars, both from a volumetric and gravimetric point of view, followed by a description of the best approved methods of the direct precipitation of sugars by such reagents as barium hydroxid and phenylhydrazin.