(A) = Milligrams of copper.
(B) = Milligrams of levulose.
(A) (B) (A) (B) (A) (B) (A) (B)
20 7.15 62 31.66 104 56.85 146 82.81
21 7.78 63 32.25 105 57.46 147 83.43
22 8.41 64 32.84 106 58.07 148 84.06
23 9.04 65 33.43 107 58.68 149 84.68
24 9.67 66 34.02 108 59.30 150 85.31
25 10.30 67 34.62 109 59.91 151 85.93
26 10.81 68 35.21 110 60.52 152 86.55
27 11.33 69 35.81 111 61.13 153 87.16
28 11.84 70 36.40 112 61.74 154 87.88
29 12.36 71 37.00 113 62.36 155 88.40
30 12.87 72 37.59 114 62.97 156 89.05
31 13.46 73 38.19 115 63.58 157 89.69
32 14.05 74 38.78 116 64.21 158 90.34
33 14.64 75 39.38 117 64.84 159 90.98
34 15.23 76 39.98 118 65.46 160 91.63
35 15.82 77 40.58 119 66.09 161 92.26
36 16.40 78 41.17 120 66.72 162 92.90
37 16.99 79 41.77 121 67.32 163 93.53
38 17.57 80 42.37 122 67.92 164 94.17
39 18.16 81 42.97 123 68.53 165 94.80
40 18.74 82 43.57 124 69.13 166 95.44
41 19.32 83 44.16 125 69.73 167 96.08
42 19.91 84 44.76 126 70.35 168 96.77
43 20.49 85 45.36 127 70.96 169 97.33
44 21.08 86 45.96 128 71.58 170 97.99
45 21.66 87 46.57 129 72.19 171 98.63
46 22.25 88 47.17 130 72.81 172 99.27
47 22.83 89 47.78 131 73.43 173 99.90
48 23.42 90 48.38 132 74.05 174 100.54
49 24.00 91 48.98 133 74.67 175 101.18
50 24.59 92 49.58 134 75.29 176 101.82
51 25.18 93 50.18 135 75.91 177 102.46
52 25.76 94 50.78 136 76.53 178 103.11
53 26.35 95 51.38 137 77.15 179 103.75
54 26.93 96 51.98 138 77.77 180 104.39
55 27.52 97 52.58 139 78.39 181 105.04
56 28.11 98 53.19 140 79.01 182 105.68
57 28.70 99 53.79 141 79.64 183 106.33
58 29.30 100 54.39 142 80.28 184 106.97
59 29.89 101 55.00 143 80.91 185 107.62
60 30.48 102 55.62 144 81.55 186 108.27
61 31.07 103 56.23 145 82.18 187 108.92
(A) (B) (A) (B) (A) (B) (A) (B)
188 109.56 232 138.57 276 168.68 320 199.97
189 110.21 233 139.25 277 169.37 321 200.71
190 110.86 234 139.18 278 170.06 322 201.44
191 111.50 235 140.59 279 170.75 323 202.18
192 112.14 236 141.27 280 171.44 324 202.91
193 112.78 237 141.94 281 172.14 325 203.65
194 113.42 238 142.62 282 172.85 326 204.39
195 114.06 239 143.29 283 173.55 327 205.13
196 114.72 240 143.97 284 174.26 328 205.88
197 115.38 241 144.65 285 174.96 329 206.62
198 116.04 242 145.32 286 175.67 330 207.36
199 116.70 243 146.00 287 176.39 331 208.10
200 117.36 244 146.67 288 177.10 332 208.83
201 118.02 245 147.35 289 177.82 333 209.57
202 118.68 246 148.03 290 178.53 334 210.30
203 119.33 247 148.71 291 179.24 335 211.04
204 119.99 248 149.40 292 179.95 336 211.78
205 120.65 249 150.08 293 180.65 337 212.52
206 121.30 250 150.76 294 181.63 338 213.25
207 121.96 251 151.44 295 182.07 339 213.99
208 122.61 252 152.12 296 182.78 340 214.73
209 123.27 253 152.81 297 183.49 341 215.48
210 123.92 254 153.49 298 184.21 342 216.23
211 124.58 255 154.17 299 184.92 343 216.97
212 125.24 256 154.91 300 185.63 344 217.72
213 125.90 257 155.65 301 186.35 345 218.47
214 126.56 258 156.40 302 187.06 346 219.21
215 127.22 259 157.14 303 187.78 347 219.97
216 127.85 260 157.88 304 188.49 348 220.71
217 128.48 261 158.49 305 189.21 349 221.46
218 129.10 262 159.09 306 189.93 350 222.21
219 129.73 263 159.70 307 190.65 351 222.96
220 130.36 264 160.30 308 191.37 352 223.72
221 131.07 265 160.91 309 192.09 353 224.47
222 131.77 266 161.63 310 192.81 354 225.23
223 132.48 267 162.35 311 193.53 355 225.98
224 133.18 268 163.07 312 194.25 356 226.74
225 133.89 269 163.79 313 194.97 357 227.49
226 134.56 270 164.51 314 195.69 358 228.25
227 135.23 271 165.21 315 196.41 359 229.00
228 135.89 272 165.90 316 197.12 360 229.76
229 136.89 273 166.60 317 197.83 361 230.52
230 137.23 274 167.29 318 198.55 362 231.28
231 137.90 275 167.99 319 199.26 363 232.05
(A) (B) (A) (B) (A) (B) (A) (B)
364 232.81 370 237.39 376 241.87 382 246.25
365 233.57 371 238.16 377 242.51 383 247.17
366 234.33 372 238.93 378 243.15 384 248.08
367 235.10 373 239.69 379 243.79 385 248.99
368 235.86 374 240.46 380 244.43
369 236.63 375 241.23 381 245.34
=147. Precipitation of Sugars with Phenylhydrazin=.—The combination of
phenylhydrazin with aldehyds and ketones was first studied by Fischer,
and the near relationship of these bodies to sugar soon led to the
investigation of the compounds formed thereby with this reagent.[112]
Reducing sugars form with phenylhydrazin insoluble crystalline bodies,
to which the name osazones has been given. The reaction which takes
place is a double one and is represented by the following formulas:
Dextrose. Phenylhydrazin. Dextrose-phenylhydrazone.
C₆H₁₂O₆ + C₆H₅NH.NH₂ = C₆H₁₂O₅.N.NHC₆H₅ + H₂O
and C₆H₁₂O₅.N.NHC₆H₅ + C₆H₅NH.NH₂ =
Phenyldextrosazone.
C₆H₁₀O₄(N.NHC₆H₅)₂ + 2H₂O.
The dextrosazone is commonly called glucosazone. The osazones formed
with the commonly occurring reducing sugars are crystalline, stable,
insoluble bodies which can be easily separated from any attending
impurities and identified by their melting points. Glucosazone melts at
205°, lactosazone at 200° and maltosazone at 206°.
The osazones are precipitated in the following way: The reducing sugar,
in about ten per cent solution, is treated with an excess of the
acetate of phenylhydrazin in acetic acid and warmed to from 75° to 85°.
In a short time the separation is complete and the yellow precipitate
formed is washed, dried and weighed. The sugar can be recovered from
the osazone by decomposing it with strong hydrochloric acid by means
of which the phenylhydrazin is displaced and a body, osone, is formed,
which by treatment with zinc dust and acetic acid, is reduced to the
original sugar. The reactions which take place are represented by the
following equations:[113]
Glucososone.
C₆H₁₀O₄(N.NH.C₆H₅)₂ + 2H₂O = C₆H₁₀O₆ + 2C₆H₅N₂H₂
Dextrose (Glucose).
C₆H₁₀O₆ + H₂ = C₆H₁₂O₆.
For the complete precipitation of dextrose as osazone Lintner and
Kröber show that the solution of dextrose should not contain more than
one gram in 100 cubic centimeters. Twenty cubic centimeters containing
0.2 gram dextrose should be used for the precipitation.[114] To this
solution should be added one gram of phenylhydrazin and one gram of
fifty per cent acetic acid. The solution is then to be warmed for about
two hours and the precipitate washed with from sixty to eighty cubic
centimeters of hot water and dried for three hours at 105°. One part
of the osazone is equivalent to one part of dextrose when maltose and
dextrin are absent. When these are present the proportion is one part
of osazone to 1.04 of dextrose. Where levulose is precipitated instead
of dextrose 1.43 parts of the osazone are equal to one part of the
sugar.
Sucrose is scarcely at all precipitated as osazone until inverted.
After inversion and precipitation as above, 1.33 parts osazone are
equal to one part of sucrose.
The reaction with phenylhydrazin has not been much used for quantitive
estimations of sugars, but it has been found especially useful in
identifying and separating reducing sugars. It is altogether probable,
however, that in the near future phenylhydrazin will become a common
reagent for sugar work.
Maquenne has studied the action of phenylhydrazin on sugars and
considers that this reaction offers the only known means of
precipitating these bodies from solutions where they are found mixed
with other substances.[115] The osazones, which are thus obtained, are
usually very slightly soluble in the ordinary reagents, for which
reason it is easy to obtain them pure when there is at the disposition
of the analyst a sufficient quantity of the material. But if the sugar
to be studied is rare and if it contain, moreover, several distinct
reducing bodies, the task is more delicate. It is easy then to confound
several osazones which have almost identical points of fusion;
for example, glucosazone with galactosazone. Finally, it becomes
impossible by the employment of phenylhydrazin to distinguish glucose,
dextrose or mannose from levulose alone or mixed with its isomers.
Indeed, these three sugars give, with the acetate of phenylhydrazin
the same phenylglucosazone which melts at about 205°. It is noticed
that the weights of osazones which are precipitated when different
sugars are heated for the same time with the same quantity of the
phenylhydrazin, vary within extremely wide limits. It is constant for
each kind of sugar if the conditions under which the precipitation
is made are rigorously the same. There is then, in the weight of the
osazones produced, a new characteristic of particular value. The
following numbers have been obtained by heating for one hour at 100°,
one gram of sugar with 100 cubic centimeters of water and five cubic
centimeters of a solution containing forty grams of phenylhydrazin
and forty grams of acetic acid per hundred. After cooling the liquid,
the osazones are received upon a weighed filter, washed with 100
cubic centimeters of water, dried at 110° and weighed. The weights of
osazones obtained are given in the following table:
Weight of the osazones.
Character of the sugar. gram.
Sorbine, crystallized 0.82
Levulose ” 0.70
Xylose ” 0.40
Glucose, anhydrous 0.32
Arabinose, crystallized 0.27
Galactose ” 0.23
Rhamnose ” 0.15
Lactose ” 0.11
Maltose ” 0.11
With solutions twice as dilute as those above, the relative conditions
are still more sensible, and the different sugars arrange themselves in
the same order, with the exception of levulose, which shows a slight
advantage over sorbine and acquires the first rank. From the above
determinations, it is shown that levulose and sorbine give vastly
greater quantities of osazones, under given conditions, than the other
reducing sugars. It would be easy, therefore, to distinguish them by
this reaction and to recognize their presence also even in very complex
mixtures, where the polarimetric examination alone would furnish only
uncertain indications.
It is remarkable that these two sugars are the only ones among the
isomers or the homologues of dextrose, actually known, which possess
the functions of an acetone. They are not, however, easily confounded,
since the glucosazone forms beautiful needles which are ordinarily
visible to the naked eye, while the sorbinosazone is still oily and
when heated never gives perfectly distinct crystals.
This method also enables us to distinguish between dextrose and
galactose, of which the osazone is well crystallized and melts at
almost the same temperature as the phenylglucosazone. Finally, it
is observed that the reducing sugars give less of osazones than the
sugars which are not capable of hydrolysis, and consequently differ
in their inversion products. It is specially noticed in this study of
the polyglucoses (bioses, trioses), that this new method of employing
the phenylhydrazin appears very advantageous. It is sufficient to
compare the weights of the osazones to that which is given under the
same conditions by a known glucose, in order to have a very certain
verification of the probabilities of the result of the chemical or
optical examination of the mixture which is under study. All the
polyglucoses which have been examined from this point of view give
very decided results. The numbers which follow have reference to one
gram of sugar completely inverted by dilute sulfuric acid, dissolved
in 100 cubic centimeters of water, and treated with two grams of
phenylhydrazin, the same quantity of acetic acid, and five grams of
crystallized sodium acetate. All these solutions have been compared
with the artificial mixtures and corresponding glucoses, with the same
quantities of the same reagents. The following are the results of the
examination:
Weight of the osazone.
Character of the sugar. gram.
1 {Saccharose, ordinary 0.71
{Glucose and levulose (.526 g each) 0.73
2 {Maltose 0.55
{Glucose (1.052 g) 0.58
3 {Raffinose, crystallized 0.48
{Levulose, glucose and galactose (.333 g each) 0.53
4 {Lactose, crystallized 0.38
{Glucose and galactose (.500 g each) 0.39
It is noticed that the agreement for each saccharose is as
satisfactory as possible. Numbers obtained with the products of
inversion are always a little low by reason of the destructive action
of sulfuric acid, and in particular, upon levulose. This is, moreover,
quite sensible when the product has to be heated for a long time with
sulfuric acid in order to secure a complete inversion. It is evident
from the data cited from the papers of Fischer, Maquenne, and others,
that the determination of sugars by this method is not a very difficult
analytical process and may, in the near future, become of great
practical importance.
=148. Molecular Weights of Carbohydrates.=—In the examination of
carbohydrates the determination of the molecular weights is often of
the highest analytical value.
The uncertainty in respect of the true molecular weights of the
carbohydrates is gradually disappearing by reason of the insight into
the composition of these bodies, which recently discovered physical
relations have permitted.
Raoult, many years ago,[116] proposed a method of determining molecular
weights which is particularly applicable to carbohydrates soluble in
water.
The principle of Raoult’s discovery may be stated as follows: The
depression of the freezing point of a liquid, caused by the presence of
a dissolved liquid or solid, is proportionate to the absolute amount of
substance dissolved and inversely proportionate to its molecular weight.
The following formulas may be used in computing results:
_C_ = observed depression of freezing point:
_P_ = weight of anhydrous substance in 100 grams:
_C_
--- = _A_ = depression produced by one gram substance in 100 grams:
_P_
_K_ = depression produced by dissolving in 100 cubic centimeters a
number of grams of the substance corresponding to its molecular weight:
_M_ = molecular weight:
_C_
Then we have, _K_ = ---- × _M_.
_P_
_K_ is a quantity varying with the nature of the solvent but with
the same solvent remaining sensibly constant for numerous groups of
compounds.