Part 16

In the conduct of the work twenty-five cubic centimeters of the copper solution are mixed with an equal quantity of the alkaline tartrate mixture, and from twenty to one hundred cubic centimeters of the sugar solution added, according to its concentration. This solution should not contain less than seventy nor more than 306 milligrams of lactose. The volume is completed to 150 cubic centimeters with boiling water and kept in lively ebullition for six minutes. The rest of the operation is conducted in the manner already described. From the weight of copper obtained the quantity of milk sugar is determined by inspecting the table. It is recommended to use such a weight of milk sugar as will give about 200 milligrams of copper.

TABLE FOR DETERMINING MILK SUGAR.

(A) = Milligrams of copper. (B) = Milligrams of milk sugar.

(A) (B) (A) (B) (A) (B) (A) (B) 100 71.6 120 86.4 140 101.3 160 116.4 101 72.4 121 87.2 141 102.0 161 117.1 102 73.1 122 87.9 142 102.8 162 117.9 103 73.8 123 88.7 143 103.5 163 118.6 104 74.6 124 89.4 144 104.3 164 119.4 105 75.3 125 90.1 145 105.1 165 120.2 106 76.1 126 90.9 146 105.8 166 120.9 107 76.8 127 91.6 147 106.6 167 121.7 108 77.6 128 92.4 148 107.3 168 122.4 109 78.3 129 93.1 149 108.1 169 123.2 110 79.0 130 93.8 150 108.8 170 123.9 111 79.8 131 94.6 151 109.6 171 124.7 112 80.5 132 95.3 152 110.3 172 125.5 113 81.3 133 96.1 153 111.1 173 126.2 114 82.0 134 96.9 154 111.9 174 127.0 115 82.7 135 97.6 155 112.6 175 127.8 116 83.5 136 98.3 156 113.4 176 128.5 117 84.2 137 99.1 157 114.1 177 129.3 118 85.0 138 99.8 158 114.9 178 130.1 119 85.7 139 100.5 159 115.6 179 130.8

(A) (B) (A) (B) (A) (B) (A) (B) 180 131.6 223 164.2 266 197.2 309 231.4 181 132.4 224 164.9 267 198.0 310 232.2 182 133.1 225 165.7 268 198.8 311 232.9 183 133.9 226 166.4 269 199.5 312 233.7 184 134.7 227 167.2 270 200.3 313 234.5 185 135.4 228 167.9 271 201.1 314 235.3 186 136.2 229 168.6 272 201.9 315 236.1 187 137.0 230 169.4 273 202.7 316 236.8 188 137.7 231 170.1 274 203.5 317 237.6 189 138.5 232 170.9 275 204.3 318 238.4 190 139.3 233 171.6 276 205.1 319 239.2 191 140.0 234 172.4 277 205.9 320 240.0 192 140.8 235 173.1 278 206.7 321 240.7 193 141.6 236 173.9 279 207.5 322 241.5 194 142.3 237 174.6 280 208.3 323 242.3 195 143.1 238 175.4 281 209.1 324 243.1 196 143.9 239 176.2 282 209.9 325 243.9 197 144.6 240 176.9 283 210.7 326 244.6 198 145.4 241 177.7 284 211.5 327 245.4 199 146.2 242 178.5 285 212.3 328 246.2 200 146.9 243 179.3 286 213.1 329 247.0 201 147.7 244 180.1 287 213.9 330 247.7 202 148.5 245 180.8 288 214.7 331 248.5 203 149.2 246 181.6 289 215.5 332 249.2 204 150.0 247 182.4 290 216.3 333 250.0 205 150.7 248 183.2 291 217.1 334 250.8 206 151.5 249 184.0 292 217.9 335 251.6 207 152.2 250 184.8 293 218.7 336 252.5 208 153.0 251 185.5 294 219.5 337 253.3 209 153.7 252 186.3 295 220.3 338 254.1 210 154.5 253 187.1 296 221.1 339 254.9 211 155.2 254 187.9 297 221.9 340 255.7 212 156.0 255 188.7 298 222.7 341 256.5 213 156.7 256 189.4 299 223.5 342 257.4 214 157.5 257 190.2 300 224.4 343 258.2 215 158.2 258 191.0 301 225.2 344 259.0 216 159.0 259 191.8 302 225.9 345 259.8 217 159.7 260 192.5 303 226.7 346 260.6 218 160.4 261 193.3 304 227.5 347 261.4 219 161.2 262 194.1 305 228.3 348 262.3 220 161.9 263 194.9 306 229.1 349 263.1 221 162.7 264 195.7 307 229.8 350 263.9 222 163.4 265 196.4 308 230.6 351 264.7

(A) (B) (A) (B) (A) (B) (A) (B) 352 265.5 365 276.2 377 286.5 389 296.8 353 266.3 366 277.1 378 287.4 390 297.7 354 267.2 367 277.9 379 288.2 391 298.5 355 268.0 368 278.8 380 289.1 392 299.4 356 268.8 369 279.6 381 289.9 393 300.3 357 269.6 370 280.5 382 290.8 394 301.1 358 270.4 371 281.4 383 291.7 395 302.0 359 271.2 372 282.2 384 292.5 396 302.8 360 272.1 373 283.1 385 293.4 397 303.7 361 272.9 374 283.9 386 294.2 398 304.6 362 273.7 375 284.8 387 295.1 399 305.4 363 274.5 376 285.7 388 296.0 400 306.3 364 275.3

=144. Table for the Determination of Maltose.=—The copper and alkaline solutions employed for the oxidation of maltose are the same as those used for invert and milk sugars.

In the manipulation twenty-five cubic centimeters each of the copper and alkali solutions are mixed and boiled and an equal volume of the maltose solution added, which should not contain more than one per cent of the sugar. The boiling is continued for four minutes, an equal volume of cold recently boiled water added, the cuprous oxid separated by filtration and the metallic copper obtained in the manner already described. The weight of maltose oxidized is then ascertained from the table.

_Example._ Weight of impure maltose taken, ten grams to a liter: Quantity used, twenty-five cubic centimeters: Weight of copper obtained 268 milligrams: Weight of maltose oxidized 237 milligrams: Weight of impure maltose taken 250 milligrams: Percentage of maltose in sample 94.8.

TABLE FOR MALTOSE.

(A) = Milligrams of copper. (B) = Milligrams of maltose.

(A) (B) (A) (B) (A) (B) (A) (B) 30 25.3 35 29.6 40 33.9 45 38.3 31 26.1 36 30.5 41 34.8 46 39.1 32 27.0 37 31.3 42 35.7 47 40.0 33 27.9 38 32.2 43 36.5 48 40.9 34 28.7 39 33.1 44 37.4 49 41.8

(A) (B) (A) (B) (A) (B) (A) (B) 50 42.6 94 81.2 138 120.6 182 160.1 51 43.5 95 82.1 139 121.5 183 160.9 52 44.4 96 83.0 140 122.4 184 161.8 53 45.2 97 83.9 141 123.3 185 162.7 54 46.1 98 84.8 142 124.2 186 163.6 55 47.0 99 85.7 143 125.1 187 164.5 56 47.8 100 86.6 144 126.0 188 165.4 57 48.7 101 87.5 145 126.9 189 166.3 58 49.6 102 88.4 146 127.8 190 167.2 59 50.4 103 89.2 147 128.7 191 168.1 60 51.3 104 90.1 148 129.6 192 169.0 61 52.2 105 91.0 149 130.5 193 169.8 62 53.1 106 91.9 150 131.4 194 170.7 63 53.9 107 92.8 151 132.3 195 171.6 64 54.8 108 93.7 152 133.2 196 172.5 65 55.7 109 94.6 153 134.1 197 173.4 66 56.6 110 95.5 154 135.0 198 174.3 67 57.4 111 96.4 155 135.9 199 175.2 68 58.3 112 97.3 156 136.8 200 176.1 69 59.2 113 98.1 157 137.7 201 177.0 70 60.1 114 99.0 158 138.6 202 177.9 71 61.0 115 99.9 159 139.5 203 178.7 72 61.8 116 100.8 160 140.4 204 179.6 73 62.7 117 101.7 161 141.3 205 180.5 74 63.6 118 102.6 162 142.2 206 181.4 75 64.5 119 103.5 163 143.1 207 182.3 76 65.4 120 104.4 164 144.0 208 183.2 77 66.2 121 105.3 165 144.9 209 184.1 78 67.1 122 106.2 166 145.8 210 185.0 79 68.0 123 107.1 167 146.7 211 185.9 80 68.9 124 108.0 168 147.6 212 186.8 81 69.7 125 108.9 169 148.5 213 187.7 82 70.6 126 109.8 170 149.4 214 188.6 83 71.5 127 110.7 171 150.3 215 189.5 84 72.4 128 111.6 172 151.2 216 190.4 85 73.2 129 112.5 173 152.0 217 191.2 86 74.1 130 113.4 174 152.9 218 192.1 87 75.0 131 114.3 175 153.8 219 193.0 88 75.9 132 115.2 176 154.7 220 193.9 89 76.8 133 116.1 177 155.6 221 194.8 90 77.7 134 117.0 178 156.5 222 195.7 91 78.6 135 117.9 179 157.4 223 196.6 92 79.5 136 118.8 180 158.3 224 197.5 93 80.3 137 119.7 181 159.2 225 198.4

(A) (B) (A) (B) (A) (B) (A) (B) 226 199.3 245 216.3 264 233.4 283 250.4 227 200.2 246 217.2 265 234.3 284 251.3 228 201.1 247 218.1 266 235.2 285 252.2 229 202.0 248 219.0 267 236.1 286 253.1 230 202.9 249 219.9 268 237.0 287 254.0 231 203.8 250 220.8 269 237.9 288 254.9 232 204.7 251 221.7 270 238.8 289 255.8 233 205.6 252 222.6 271 239.7 290 256.6 234 206.5 253 223.5 272 240.6 291 257.5 235 207.4 254 224.4 273 241.5 292 258.4 236 208.3 255 225.3 274 242.4 293 259.3 237 209.1 256 226.2 275 243.3 294 260.2 238 210.0 257 227.1 276 244.2 295 261.1 239 210.9 258 228.0 277 245.1 296 262.0 240 211.8 259 228.9 278 246.0 297 262.8 241 212.7 260 229.8 279 246.9 298 263.7 242 213.6 261 230.7 280 247.8 299 264.6 243 214.5 262 231.6 281 248.7 300 265.5 244 215.4 263 232.5 282 249.6

=145. Preparation of Levulose.=—It is not often that levulose, unmixed with other reducing sugars, is brought to the attention of the analyst. It probably does not exist in the unmixed state in any agricultural product. The easiest method of preparing it is by the hydrolysis of inulin. A nearly pure levulose has also lately been placed on the market under the name of diabetin. It is prepared from invert sugar.

Inulin is prepared from dahlia bulbs by boiling the pulp with water and a trace of calcium carbonate. The extract is concentrated to a sirup and subjected to a freezing temperature to promote the crystallization of the inulin. The separated product is subjected to the above operations several times until it is pure and colorless. It is then washed with alcohol and ether and is reduced to a fine powder. Before the repeated treatment with water it is advisable to clarify the solution with lead subacetate. The lead is afterwards removed by hydrogen sulfid and the resultant acetic acid neutralized with calcium carbonate.

By the action of hot dilute acids inulin is rapidly converted into levulose.

Levulose may also be prepared from invert sugar, but in this case it is difficult to free it from traces of dextrose. The most successful method consists in forming a lime compound with the invert sugar and separating the lime levulosate and dextrosate by their difference in solubility. The levulose salt is much less soluble than the corresponding compound of dextrose. In the manufacture of levulose from beet molasses, the latter is dissolved in six times its weight of water and inverted with a quantity of hydrochloric acid, proportioned to the quantity of ash present in the sample. After inversion the mixture is cooled to zero and the levulose precipitated by adding fine-ground lime. The dextrose and coloring matters in these conditions are not thrown down. The precipitated lime levulosate is separated by filtration and washed with ice-cold water. The lime salt is afterwards beaten to a cream with water and decomposed by carbon dioxid. The levulose, after filtration, is concentrated to the crystallizing point.[110]

=146. Estimation of Levulose.=—Levulose, when free of any admixture with other reducing sugars, may be determined by the copper method with the use of the subjoined table, prepared by Lehmann.[111] The copper solution is the same as that used for invert sugar, _viz._, 69.278 grams of pure copper sulfate in one liter. The alkali solution is prepared by dissolving 346 grams of rochelle salt and 250 grams of sodium hydroxid in water and completing the volume to one liter.

_Manipulation._—Twenty-five cubic centimeters of each solution are mixed with fifty of water and boiled. To the boiling mixture twenty-five cubic centimeters of the levulose solution are added, which must not contain more than one per cent of the sugar. The boiling is then continued for fifteen minutes, and the cuprous oxid collected, washed and reduced to the metallic state in the usual way. The quantity of levulose is then determined by inspection from the table given below. Other methods of determining levulose in mixtures will be given further on.

TABLE FOR THE ESTIMATION OF LEVULOSE.

(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: