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]

for raffinose the formula would be

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

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

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.

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:

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

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 a_D.[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:

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:

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.

VOLUMETRIC METHODS.

109. Classification.—Among the volumetric methods will be given those which are in common use or such as have been approved by the practice of analysts. Since the use of mercuric salts is now practiced to a limited extent, only a brief study of that process will be attempted. With the copper methods a somewhat extended description will be given of those depending on the use of copper sulfate, and a briefer account of the copper carbonate process.

In the copper sulfate method two distinct divisions must be noted, viz., first an indirect process depending first upon the reduction of the copper to a suboxid, the subsequent action of this body on iron salts, measured finally by titration with potassium permanganate; and second, a direct process determined either by the disappearance of the blue color from the copper solution, or by the absence of copper from a drop of the solution withdrawn and tested with potassium ferrocyanid. This last mentioned reaction is one which is found in common use. The volumetric methods are not, as a rule, as accurate as the gravimetric, depending on weighing the resultant metal, but they are far more rapid and well suited to technical control determinations.

110. Reduction of Mercuric Salts.—The method of determining sugar by its action on mercuric salts, is due to Knapp.[70] The method is based on the observation that dextrose and other allied sugars, will reduce an alkaline solution of mercuric cyanid, and that the mercury will appear in a metallic state.

The mercuric liquor is prepared by adding to a solution of ten grams of mercuric cyanid, 100 cubic centimeters of a solution of caustic soda of 1.145 specific gravity, and making the volume to one liter with water. The solution of sugar to be titrated, should be as nearly as possible of one per cent strength.

To 100 cubic centimeters of the boiling solution, the sugar solution is added in small portions from a burette and in such a way as to keep the whole mass in gentle ebullition.

To determine when all the mercuric salt has been decomposed, a drop of the clear boiling liquid is removed and brought into contact with a drop of stannous chlorid solution on a white surface. A brownish black coloration or precipitate will indicate that the mercury is not all precipitated. Fresh portions of the sugar must then be added, until no further indication of the presence of mercury is noted. The approximate quantity of sugar solution required to precipitate the mercury having thus been determined, the process is repeated by adding rapidly, nearly the quantity of sugar solution required, and then only a few drops at a time, until the reduction is complete.

One hundred cubic centimeters of the mercuric cyanid solution prepared as directed above, will be completely reduced by

By reason of the unpleasant odor of the boiling mercuric cyanid when in presence of a reducing agent, the process should be conducted in a well ventilated fume chamber. With a little practice the process is capable of rapid execution, and gives reasonably accurate results.

111. Sachsse’s Solution.—The solution of mercuric salts proposed by Sachsse, is made by dissolving eighteen grams of mercuric iodid in twenty-five cubic centimeters of an aqueous solution of potassium iodid. To this solution are added 200 cubic centimeters of potash lye, containing eighty grams of caustic potash. After mixing the solution, the volume is completed to one liter. The sugar solutions used to reduce this mixture, should be more dilute than those employed with the mercuric cyanid, and should not be over one-half per cent in strength. The end of the reduction is determined as already described. After a preliminary trial, nearly all the sugar necessary to complete reduction, should be added at once, and the end of the reduction then determined by the addition of successive small quantities. One hundred cubic centimeters of the mercuric iodid solution prepared as directed above, require the following quantities of sugar to effect a complete reduction:

By reason of the great difference between the reducing power of dextrose and levulose in this solution, it has been used in combination with the copper reduction method, to be described, to determine the relative proportion of dextrose and levulose in a mixture.[71]

It is now known that copper solutions require slightly different quantities of dextrose, levulose, or invert sugar to effect complete reduction, but the variations are not great and in the calculation above mentioned, it may be assumed that these differences do not exist.

Instead of using stannous chlorid as an indicator, the end of the reaction may be determined as follows: A disk of filtering paper is placed over a small beaker containing some ammonium sulfid. A drop of the clear hot solution is placed on this disk, and if salts of mercury be still present a dark stain will be produced; or a drop of the ammonium sulfid may be brought near the moist spot formed by the drop of mercury salt. An alkaline solution of zinc oxid may also be used.

The methods depending on the use of mercuric salts have, of late, been supplanted by better processes, and space will not be given here to their further discussion.

112. The Volumetric Copper Methods.—The general principle on which these methods depend, is found in the fact that certain sugars, notably, dextrose, (glucose), levulose, (fructose), maltose and lactose, have the property of reducing an alkaline solution of copper to a lower state of combination, in which the copper is separated as cuprous oxid. The end of the reaction is either determined by the disappearance of the blue color of the solution, or by the reaction produced by a drop of the hot filtered solution, when placed in contact with a drop of potassium ferrocyanid acidified with acetic.

The copper salt which is found to give the most delicate and reliable reaction, is the tartrate. The number of volumetric processes proposed and which are in use, is very great, and an attempt even to enumerate all of these can not be made in this volume. A few of the most reliable and best attested methods will be given, representing if possible, the best practice in this and other countries. The rate of reduction of the copper salt to suboxid, is influenced by the rate of mixing with the sugar solutions, the temperature, the composition of the copper solution and the strength of the sugar solution.

The degree of reduction is also modified by the rate at which the sugar solution is added, and by the degree and duration of heating, and all these variables together, make the volumetric methods somewhat difficult and their data, to a certain extent, discordant. By reason, however, of the ease with which they are applied and the speed of their execution, they are invaluable for approximately correct work and for use in technical control.

113. Historical.—It is not the purpose in this paragraph to trace the development of the copper reduction method for the determination of reducing sugars, but only to refer to the beginning of the exact analytical application of it.

Peligot, as early as 1844, made a report to the Society for the Encouragement of National Industry on methods proposed by Barreswil and Fromherz for the quantitive estimation of sugar by means of copper solution.[72] These methods were based on the property of certain sugars to reduce alkaline copper solution to a state of cuprous oxid first announced by Trommer.[73] This was followed by a paper by Falck on the quantitive determination of sugar in urine.[74]

In 1848 the methods, which have been proposed, were critically examined by Fehling, and from the date of his paper the determination of sugar by the copper method may be regarded as resting on a scientific basis.[75]

Since the date mentioned the principal improvements in the process have been in changing the composition of the copper solution in order to render it more stable, which has been accomplished by varying the proportions of copper sulfate, alkali and tartaric acid. For the better keeping of the solution the method of preserving the copper sulfate and the alkaline tartrates in separate flasks and only mixing them at the time of use has been found very efficacious.[76] For testing for the end of the reaction by means of an acetic acid solution of potassium ferrocyanid the filtering tube suggested by the author, the use of which will be described further on, has proved quite useful. Pavy has suggested that by the addition of ammonia to the copper solution the precipitated suboxid may be kept in solution and the end of the reaction thus easily distinguished by the disappearance of the blue color.[77] Allen has improved on this method by covering the hot mixture with a layer of paraffin oil whereby any oxidation of the suboxid is prevented.[78]

The introduction and development of the gravimetric process depending on securing the reduced copper oxid in a metallic state as developed by Allihn, Soxhlet, and others, completes the resumé of this brief sketch of the rise and development of the process.

114. Action of Alkaline Copper Solution on Dextrose.—The action to which dextrose and other reducing sugars are subjected in the presence of a hot alkaline copper solution is two-fold in its nature. In the first place there is an oxidation of the sugar which is transformed into tartronic, formic and oxalic acids, the two latter in very small quantities. At the same time another part of the sugar is attacked directly by the alkali and changed to complex products among which have been detected lactic, oxyphenic and oxalic acids, also two bodies isomeric with dioxyphenolpropionic acid. When the sugar is in large excess melassic and glucic acids have also been detected. The glucic acid may be regarded as being formed by simple dehydration but becomes at once resolved into pyrocatechin and gluconic acid according to the reaction C₁₂H₁₈O₉ = C₆H₆O₂ + C₅H₁₂O₇. The gluconic acid also is decomposed and gives birth to lactic and glyceric acids according to the formula C₆H₁₂O₇ = C₃H₆O₃ + C₃H₆O₄. The glyceric acid also in the presence of a base is changed into lactic and oxalic acids. Between lactic acid and pyrocatechin, existing in a free state, there is produced a double reciprocal etherification in virtue of which there arise two ethers isomeric with hydrocaffeic acid, C₉H₁₀O₄. One of these bodies is an acid and corresponds to the constitution

• CH₃
•   /
•   O ── CH
• /  \
• C₆H₄ CO₂H (2)
• \
• OH (2)

and the other is of an alcoholic nature corresponding to the formula

•  CO₂ ── CHOH ── CH₃ (1)
• /
• C₆H₄
• \
• OH₂ (2)

Of all these products only oxyphenic and lactic acids and their ethers and oxalic acid remain unchanged and they can be isolated. All the others are transformed in an acid state and they can only be detected by operating in the presence of metallic oxids capable of precipitating them at the time of their formation.[79]

115. Fehling’s Solution.—The copper solution which has been most used in the determination of reducing sugars is the one proposed by Fehling as a working modification of the original reagent used by Trommer.[80]

Following is the formula for the preparation of the fehling solution:

The copper sulfate is dissolved in water and the potassium tartrate in the aqueous solution of the sodium hydroxid which should have a volume of about 700 cubic centimeters. The two solutions are mixed and the volume completed to a liter. Each cubic centimeter of this solution will be reduced by five milligrams of dextrose, equivalent to four and a half milligrams of sucrose.

The reaction which takes place is represented by the following molecular proportions:

Fehling’s solution is delicate in its reactions but does not keep well, depositing cuprous oxid on standing especially in a warm place exposed to light. The fehling liquor was soon modified in its constitution by substituting 173 grams of the double sodium and potassium tartrate for the neutral potassium tartrate first used, and, in fact, the original fehling reagent contained forty grams of copper sulfate instead of the quantity mentioned above. Other proportions of the ingredients are also given by many authors as fehling solution.

116. Comparison of Copper Solutions for Oxidizing Sugars.—For the convenience of analysts there is given below a tabular comparison of the different forms of fehling liquor which have been proposed for oxidizing sugars. The table is based on a similar one prepared by Tollens and Rodewald, amended and completed by Horton.[81] The solutions are arranged alphabetically according to authors’ names: