Chapter XII

Carbohydrates

147. Properties of Dextrose (Sections 282-284). - (a) Taste and solubility of dextrose. - Taste equal amounts of dextrose and cane-sugar. Determine whether dextrose is soluble in the following liquids: water, alcohol, and ether.

(b) Presence of alcoholic hydroxyl groups in dextrose. - In a 100 cc. bottle shake 10 cc. of a 10 per cent solution of dextrose with 2 cc. of benzoyl chloride and 25 cc. of a 10 per cent solution of sodium hydroxide. Shake vigorously until the odor of the benzoyl chloride disappears. Write the structural formula of the compound formed.

(c) Dextrose and copper hydroxide. - Precipitate copper hydroxide by adding 5 cc. of a solution of sodium hydroxide to 1 cc. of a solution of copper sulphate, and then add 5 cc. of a strong solution of dextrose (about 20 per cent) and shake. It is necessary to have a large excess of the alkali present. Compare with experiment 142f, page 108. Heat the solution nearly to boiling.

(d) Dextrose and alkalies. - Boil 10 cc. of a 10 per cent solution of dextrose with 5 cc. of a solution of sodium hydroxide. Compare the result with that obtained with aldehyde and sodium hydroxide (experiment 108d, page 83).

(e) Dextrose and Schiff's reagent. - Determine whether dextrose produces a color with Schiff's reagent (experiment 108h, page 83).

(f) Reducing action of dextrose: silver mirror. - Test a dilute solution of glucose with an ammoniacal solution of silver nitrate or with Tollen's reagent (experiment 106d, page 81).

(g) Reducing action of dextrose: Fehling's solution. - Test a dilute solution of dextrose with Fehling's solution.

(h) Preparation of dextrosazone. - Dissolve 1 gram of dextrose in 20 cc. of water and add 2 grams of phenylhydrazine hydrochloride, which should be colorless (see note), and 3 grams of hydrated sodium acetate. Heat the solution by placing the flask in boiling water for 10 minutes. Cool under running water, filter by suction, and wash the crystals with cold water. Red issolve the crystals in the smallest possible amount (about 20 cc.) of boiling 50 per cent alcohol. Filter the hot solution, cool in running water, filter off the crystals, and dry them on a porous plate, and finally at 100°. Determine the melting-point of the crystals in a bath, the temperature of which is rising rapidly, as dextrosazone decomposes slowly when heated. The melting-point of dextrosazone is 204°-205°.

Notes. - (b) The insoluble compound formed is a pentabenzoate of dextrose which is produced as the result of the replacement of the hydrogen atoms of the hydroxyl groups in dextrose by the benzoyl radical present in benzoyl chloride, C₆H₅COCl. The test is an application of the Baumann-Schotten reaction (see Section 463).

(c) Polyatomic alcohols and compounds related to them, such as the sugars, dissolve the hydroxides of certain heavy metals. Compounds similar to that present in Fehling's solution (Section 252) are formed. The sugars also form soluble compounds with calcium and barium hydroxides.

(e) Although many of the carbohydrates contain the aldehyde group, they do not produce a color with Schiff's reagent.

(h) Phenylhydrazine and its salts decompose rapidly when exposed to air and to light. Pure compounds must be used in the preparation of osazones. The phenylhydrazine can be dissolved in an excess of acetic acid, just before use, or the hydrochloride and sodium acetate may be used. The pure hydrochloride can be prepared as follows: Redistil the phenylhydrazine if it is colored. Dissolve the compound in 12 times its volume of alcohol, and add pure concentrated hydrochloric acid as long as a precipitate is formed. Filter off the precipitate by suction, and wash it with alcohol until it is colorless. Wash twice with ether. If the salt is not to be used immediately, dry it in the air for half an hour, and then for 1 hour at 100°. Place the salt in a tightly stoppered bottle of brown glass.


Monosaccharides and Disaccharides

148. General Reactions and Properties of the Sugars. - All the experiments given in this section should be performed in each case with samples of dextrose, levulose, maltose, lactose, and sucrose.

(a) Molisch reaction for carbohydrates (Section 313). - Place a piece of the carbohydrate about the size of a mustard seed in 10 drops of water, and add 2 drops of a 10 per cent solution of a-naphthol in chloroform. Allow 1 cc. of pure concentrated sulphuric acid to flow, from a pipette, down the side of the inclined tube so that two layers are formed. A pipette for this purpose may be made by drawing out a piece of glass tubing to a fine opening at one end. Observe what happens in a few seconds. Shake and allow the mixture to stand 2 minutes; note the color. Dilute with water, note the change, add an excess of ammonia, and note the color.

(b) Solubility of sugars. - Determine whether the carbohydrates are soluble in water, in alcohol, and in ether. Dry test-tubes should be used when the solubilities in alcohol and ether are studied.

(c) Reduction of silver salts. - Determine whether dilute solutions of the carbohydrates give a mirror with an ammoniacal solution of silver nitrate or with Tollen's reagent. (See experiments 106d, page 81, and 108g, page 83.)

(d) Reduction of Fehling's solutions. - Dissolve about 0.1 gram of the carbohydrate in 5 cc. of water, and add 3 cc. of each of the solutions which combined make Fehling's solution. (See Appendix for the composition of this solution.) Heat to boiling.

(e) Fermentation of carbohydrates. - Fill fermentation - tubes with 10 per cent solutions of the carbohydrates. Add to each 2 cc. of a mixture made by rubbing one-quarter of a yeast-cake with 10 cc. of water. Label the tubes, and place them in a thermostat at 30°-35° until the next exercise. Test the gas formed. Which of the sugars are fermentated by common yeast?

(f) Formation of osazones. - Place in separate test-tubes 5 cc. of 2 per cent aqueous solutions of the five carbohydrates; label the tubes. Mix intimately 1.3 grams of colorless phenylhydrazine hydrochloride and 2 grams of crystallized sodium acetate. Divide the mixture into 5 equal parts and put one of the parts into each of the five tubes. Note the time and place the tubes in a beaker containing boiling water and heat for one-half hour. Shake the tubes when the salts have dissolved in order to mix the solution. Examine the tubes frequently and note the order in which the precipitates first appear. At the end of one-half hour place the tubes in a rack and see if any osazones crystallize out on cooling slowly. Make a full record of the results of the experiment. Examine all the osazones under a high-power microscope, and sketch the crystals.

(g) Summary of results. - Prepare a table in which are tabulated the results of experiments a, b, c, d, e, and f above. State how you could distinguish each one of the five sugars from the others.

149. Properties of Sucrose (Sections 296-297). - (a) Preparation of caramel. - Place about 2 grams of sucrose in a test-tube and heat it for 15 minutes in an oil-bath at 210°. Taste the product and determine if it is soluble in water.

(b) Sucrose and sulphuric acid. - Add to about 2 grams of sugar 2 cc. of water and then 5 cc. of concentrated sulphuric acid. If no marked change occurs heat gently. Repeat using dextrose instead of sucrose.

(c) Sucrose and alkalies. - Heat 5 cc. of a solution (about 10 per cent) of sucrose with a solution of sodium hydroxide. Is the solution highly colored? Compare with the results obtained with dextrose. What conclusion can be drawn from the experiment? What other tests lead to the same conclusion? (See experiment 148c, d, f, page 116.)

(d) Formation of tricalcium saccharate. - Shake 10 cc. of a 20 per cent solution of sucrose with an excess of milk of lime, which can be prepared by slaking a little quicklime and grinding the calcium hydroxide with enough water to make a thin paste. Filter the solution and heat it to boiling. (Eq.)

(e) Inversion of sucrose. - Boil for 5 minutes a solution of about 0.2 gram of sucrose in 10 cc. of water to which has been added 1 cc. of dilute hydrochloric acid. Neutralize the solution with sodium hydroxide, and test for a reducing sugar with Fehling's solution.

150. Isolation of Lactose from Milk (Section 300). - In a beaker heat 200 cc. of milk to about 50°, and add a dilute solution of acetic acid as long as a precipitate is formed (about 5 cc. of a 10 per cent solution of the acid). Stir until the casein collects into a ball; remove this and neutralize the solution with a dilute solution of sodium hydroxide. Make the solution weakly acidic by adding 2 or 3 drops of very dilute acetic acid. Heat the solution to boiling and add about 1 gram of precipitated calcium carbonate. Stir thoroughly and filter hot. Evaporate the solution to about 40 cc., cool, add 3 volumes of alcohol, and filter. Set aside the solution in a shallow dish until the next exercise. Filter off the crystals of lactose by suction and wash them with alcohol. Dry the crystals in the air. Taste the sugar.

Note. - The procedure adopted in this experiment is designed to bring about the precipitation of the proteins the milk contains. The solution is heated with calcium carbonate before evaporation in order to neutralize the free acid present, and thus largely prevent the hydrolysis of lactose which takes place when the sugar is heated with water in the presence of acids.

151. Oxidation of Lactose (see galactose, Section 291 and 259). - Heat 10 grams of lactose on the water-bath with about four times its weight of concentrated nitric acid (28 cc.) until the brown oxide of nitrogen is formed. Keep the mixture at 70°-80° until the evolution of gas ceases. Dilute the solution with one-half its bulk of water and let it stand until cold. Mucic and oxalic acids crystallize out. Filter, save the filtrate, and wash the crystals with warm alcohol to dissolve the oxalic acid, and then twice with a small amount of cold water. Recrystallize the residue, mucic acid, from a small amount of boiling water.

Neutralize the filtrate obtained above with solid potassium carbonate, strongly acidify with glacial acetic acid, and let stand until crystals of potassium hydrogen saccharate are formed. Filter these off and recrystallize them from the smallest possible amount of boiling water.

Dissolve a few crystals of mucic acid in a few drops of a solution of potassium hydroxide on a microscope slide. When the solution has evaporated examine the crystals under the microscope. The form of the crystals of potassium mucate is characteristic. Describe their appearance.

When the ammonium salt of saccharic acid or of mucic acid is heated, pyrrole is formed (Section 559). The latter imparts a carmine-red coloration to a pine-wood shaving which has been moistened with hydrochloric acid. Test the mucic acid obtained in this way as follows: Mix 0.1 gram of the acid with 2 cc. of ammonia and evaporate to dryness. Heat the residue strongly in a test-tube; during the heating suspend in the tube a soft pine splinter which has been soaked in concentrated hydrochloric acid for a minute or two.


Polysaccharides

152. Properties of Starches (Sections 303, 304). - (a) The form of starch grains. - Examine under the microscope and sketch the following starches: potato, arrowroot, corn, rice, and wheat.

(b) Application of the Molisch test for carbohydrates. - Test a bite of starch, filter-paper, and gum arabic as described in experiment 148a, page 115.

(c) Colloidal solutions: dialysis. - Select a piece of parchment paper, about 10 inches square, which contains no small holes. Wet the paper thoroughly and form it into a bag of about 100 cc. capacity; insert the neck of a small funnel into the mouth of the bag and fix it in place by tying a string around it. Fill the bag with water, dry it carefully on the outside, and hang it up for a few minutes. If the bag leaks it must be discarded and a new one made.

Prepare a starch solution as follows: Grind about 2 grams of starch with 10 cc. of cold water, pour the mixture into 300 cc. of boiling water, and set aside to cool. Pour the water out of the parchment bag and fill it half full with the starch solution. Take care not to spill any of the solution on the outside of the bag. Suspend the bag in a beaker containing about 100 cc. of water. The beaker should be of such a size that the water in it and that in the solution in the bags are at approximately the same level. Set aside until the next exercise and test the water outside the bag for starch according to experiment (f) below.

Prepare a second bag and carry out a similar experiment, using a 10 per cent solution of glucose. In this case test the water outside the bag for glucose by Fehling's solution. Explain dialysis and state why, by means of it, it is possible to separate the polysaccharides from the sugars.

(d) Starch and Fehling's solution. - Wash about 2 grams of starch by decantation twice. Shake up the residue with a little water, and pour it into 200 cc. of boiling water. Test about 1 cc. of this solution with Fehling's solution.

(e) Starch and alkalies. - Warm 5 cc. of the solution prepared in (c) with 5 cc. of a solution of sodium hydroxide. Compare the results with those obtained in the case of a monose (experiment 147d, page 114).

(f) Starch and iodine. - Add to 5 cc. of the solution of starch 1 drop of a solution of iodine in potassium iodide. Heat the solution to boiling, and then cool.

Shake up a little starch with cold water, filter and add a few, drops of iodine solution to the filtrate.

Test solutions of dextrose, sucrose, lactose, and dextrin with a very dilute solution of iodine (light straw-yellow in color).

(g) Hydrolysis of starch: with an acid. - Boil in an Erlenmeyer flask about 150 cc. of the starch solution prepared in (c) above with 10 cc. of concentrated hydrochloric acid. Every 5 minutes pour out about 2 cc. of the solution, cool, and test with a very dilute iodine solution. Describe the colors produced as the hydrolysis progresses. When iodine produces no color in the solution, neutralize about 5 cc. of it and test with Fehling's solution.

(h) Hydrolysis of starch: with saliva. - A free flow of saliva is easily obtained by chewing some insoluble substance such as paraffin. Collect about 40 cc. in this way. Filter the saliva through a wet filter. Test a portion of the saliva by allowing a piece of red and of blue litmus paper to stay in contact with it for 5 minutes.

Prepare 600 cc. of starch paste, using 25 grams of arrowroot starch. (Do this in the usual way, by rubbing the starch with a little cold water and pouring the suspension into boiling water. Boil for 2 or 3 minutes.) Save one-third of the paste for subsequent experiments. When the paste has cooled to 40°, mix two-thirds of it with 25 cc. of the filtered saliva and watch carefully for changes in consistency and opalescence of the mixture. At 2-minute intervals remove a few drops to a porcelain plate, and test with a very dilute iodine solution. Record the time required for the solution to clear and to reach the point where it no longer produces a color with iodine. Compare your figures with those of your neighbors. Are the salivas equally active?

Evaporate the solution in a casserole to about 109 cc. on the steam-bath. (If this point is not reached at the end of the exercise, leave the labeled casserole on the steam-bath in the care of the instructor.) Filter out the small precipitate of carbonate and cellulose. Pour the solution into 3 volumes of alcohol to precipitate dextrins. Is the amount large? Filter. Evaporate the alcohol and water on the steam-bath to a volume of about 50 cc. What is the consistency of the residue? Try the reducing power and make an osazone. What is the final product of salivary digestion?

153. Conditions Influencing Salivary Digestion. - For the following experiments dilute the filtered saliva with 5 volumes of water and dilute the starch paste with an equal volume of water.

(a) Temperature. - In each of four test-tubes put 5 cc. of starch paste. Keep two tubes at room temperature, chill the third in ice-water, and warm the fourth in a 40° water-bath. When the tubes have reached the temperature indicated, add to the second, third, and fourth in quick succession 1 cc. each of diluted saliva, and to the first 1 cc. of boiled, diluted saliva. Maintain the tubes at their respective temperatures. Note the time required for each to clear. Apply the iodine test at once and at intervals of 5 minutes. After half an hour put the first and third tubes in the 40° bath and continue the observations. Compare the effect of high and low temperature on the enzyme.

(b) Acid and alkali. - Neutralize to litmus 5 cc. of filtered saliva, using 0.4 per cent hydrochloric acid, and dilute with 5 volumes of water. Prepare a series of eight tubes containing 5 cc. each of hydrochloric acid of various percentage strengths: 0.2, 0.1, 0.05, 0.025, etc. To obtain these dilutions, measure 10 cc. of 0.2 per cent acid in a graduate, pour half into the first tube, fill the graduate to the 10 cc. mark with water, mix, pour half into the second tube, and so on. To each tube add 5 cc. of starch paste, mix thoroughly, and add to each in quick succession 1 cc. of the neutralized saliva. Apply the same tests of digestion as were used in previous experiments. What concentration of acid is inhibitory? Compare your results with those of your neighbors. Test each tube with litmus, and with congo paper.

Plan and carry out a similar experiment to determine the effect of alkali on ptyalin. Use 1 per cent sodium carbonate for the highest concentration of alkali.

(c) Condition of starch. - Test the digestibility of raw starch. Continue the experiment, using a little toluene for antiseptic, till the following exercise. What do you infer as to the desirability of cooking starchy foods thoroughly?

(d) Specificity. - Try the action of saliva on cane-sugar and Irish moss. What criterion of digestion will you use?

154. Preparation and Properties of Dextrin (Section 305). - Heat about 5 grams of starch for one-half hour at 220°-225° an oil-bath or air-bath. Pour the product into a mortar, add 2 cc. of water, and notice the adhesive quality of the mixture. Add 25 cc. of water and grind with a pestle. If there is a residue of starch which has not been converted into dextrin, filter through a folded filter. Use 2 cc. of the solution to determine whether the product reduces Fehling's solution. Test 2 cc. of the solution with a drop of iodine solution and note the color.

Add to the rest of the solution three times its volume of alcohol. Filter off the precipitated dextrin and wash twice with alcohol. Dissolve a little of the precipitate and test its reducing power with Fehling's solution. Determine the color produced by iodine solution. How does the color compare with that obtained during the intermediate stages of the hydrolysis of starch by acid (experiment 152g, page 120).

155. Properties of Cellulose (Sections 308-310). - (a) Solubility in Schweitzer's reagent. - This reagent is a saturated solution of copper hydroxide in a concentrated solution of ammonium hydroxide. It can be prepared as follows: Dissolve 5 grams of copper sulphate in about 100 cc. of water, and add a solution of sodium hydroxide as long as a precipitate is formed. Wash the precipitate three times by decantation with 500 cc. of water, and then filter through cotton; wash until the wash water is free from sulphates. Press out as much water as possible from the precipitate. Add to 10 cc. of concentrated ammonia (sp. gr. 0.90) the copper hydroxide as long as it dissolves. Add pieces of filter paper to the reagent as long as they dissolve, and filter through glass wool. Pour the solution into dilute hydrochloric acid.

(b) Preparation of parchment-paper. - Pour slowly with stirring 50 cc. of concentrated sulphuric acid into 30 cc. of water. Cool the solution to 15° to 20° and immerse in it strips of dry filter-paper. At the end of 15 to 20 seconds remove the paper, and wash it rapidly in running water; immerse the paper in a dilute solution of ammonia, and wash again with water. Test the toughness of the paper and of a piece of wet filter-paper. Put a drop of a dilute solution of iodine in potassium iodide on the paper. Hang up a piece of the paper to dry and examine it at the next exercise.

(c) Hydrolysis of cellulose. - Grind in a mortar a pinch of cotton-wool or a filter-paper with a few drops of concentrated sulphuric acid until a sticky mass is obtained; add 50 cc. of water cautiously, and boil the resulting solution for 15 minutes. Neutralize the solution with sodium hydroxide, and test for a reducing sugar with Fehling's solution.

(d) Preparation of cellulose acetate (Section 309). - In a small flask place 20 cc. of glacial acetic acid, 3 cc. of acetic anhydride, 4 drops of pure concentrated sulphuric acid, and 0.5 gram of cotton-wool. Press the cotton into the solution, and after a few minutes stir it so that most of the air bubbles are removed. Close the flask with a cork and let it stand over night or longer. (Eq.) Pour the solution in a thin stream, and with stirring, into 500 cc. of water. Filter, using a large funnel. Wrap the cellulose acetate in a piece of cotton cloth (a towel), and squeeze out as much water as possible; then set it aside until dry. Put about one-half the dry product in a small beaker or test-tube and add 10 cc. of chloroform. After standing some time the acetate should pass into solution. Pour the solution onto a watch-glass and let it evaporate slowly. When the chloroform has evaporated, put some water into the watch-glass and allow it to stand for a minute or two. Lift the edge of the film and remove it slowly from the glass. Dry the film and reserve it for a future test. Test the solubility of the rest of the acetate in glacial acetic acid, in alcohol, and in ether.

(e) Preparation of cellulose nitrate. - Pour 10 cc. of concentrated sulphuric acid into 10 cc. of concentrated nitric acid. To the hot mixture add 0.5 gram of cotton-wool. At the end of 3 minutes withdraw the nitrated cotton, and remove most of the acid adhering to it by pressing it with a glass rod against the side of the beaker. Put the cotton into a large amount of cold water. Wash for a minute in running water, squeezing out the water from time to time, and set aside to dry spontaneously. Hold a small bit of the dry nitrate with tongs, and place it in a flame. Test the solubility of a small amount of the product in alcohol. Repeat using ether. In a test-tube cover some of the nitrate with a mixture made of equal volumes of alcohol and ether. At the end of a few minutes pour off the liquid onto a glass plate or watch-glass and let the solution evaporate slowly. Remove the film as in (d) above and dry. Place the edge of the film in a flame and as soon as it begins to burn remove it. Note the rate at which the film burns. Repeat using the film made from cellulose acetate. Do you observe any difference?

156. Properties of Pentosans (Sections 295, 310, 557). - (a) Tests for lignin. - Dissolve a few drops of aniline in a few drops of dilute hydrochloric acid, and dilute with 5 cc. of water. Pour one-half of the solution onto a piece of paper made from wood-pulp (newspaper) and one-half onto a paper made from linen. Repeat the tests using a solution of phloroglucinol in dilute hydrochloric acid.

(b) Hydrolysis of pentosans. - Boil a little gum arabic or wheat bran with 10 cc. dilute hydrochloric acid, and hold over the tube a piece of paper which has been dipped into a dilute solution of aniline in acetic acid.