QUANTITATIVE TEST FOR SUGAR
Benedict’s Test.[110]—The simple quantitative test for sugar is the one devised by Benedict. This is simpler than the polariscopic examination and better suited for ordinary use.
Place 5 c.c. of Benedict’s quantitative solution in a small dish, add a little less than one-fourth of a teaspoonful of sodium carbonate and one-eighth of a teaspoonful of talcum and add 10 c.c. of water. Dilute urine (1 part urine to 9 parts water) except where the qualitative test showed a low percentage of sugar, that is, when the precipitate turns green instead of yellow, in which case it will be unnecessary to dilute the urine. Place dish over burner and bring the contents to a boil. Pour the urine into a graduated pipette. Now add the urine drop by drop to the contents in the dish until the blue color entirely disappears. This test should be done over several times to assure an accurate calculation. The calculation is made as follows: 5 c.c. of Benedict’s quantitative copper solution are reduced by 0.01 gram of glucose, consequently the quantity of undiluted urine required to reduce 5 c.c. Benedict’s solution contains 0.01 gram of glucose.
| 0.01 | × 100 = per cent. |
| x | |
| x = c.c. of undiluted urine. | |
Example; 1500 c.c. urine in 24 hours. 5 c.c. used to reduce (decolorized) Benedict’s solution.
| 0.01 | × 100 = 0.2 per cent. |
| 5 |
1500 × 0.002 (0.2 per cent.) = 3 grams of sugar in 24 hours.
Example: If the urine had been diluted with 9 parts water, in other words, 10 times, the calculation would be 5 c.c. diluted urine = 0.5 c.c. actual urine.
| 0.01 | × 100 = 2 per cent. |
| 0.5 |
1500 × 0.02 (2 per cent.) = 30 grams of sugar in 24 hours.
Hill and Eckman perform the Benedict’s quantitative test as follows:[111]
Measure with a pipette 25 c.c. Benedict’s solution into a porcelain dish, add 5 or 10 grams approximately of solid sodic carbonate, heat to boiling, and while boiling, run in urine until a white precipitate forms, then add urine more slowly until the last trace of blue disappears. The urine should be diluted so that not less than 10 c.c. will be required to amount of sugar which 25 c.c. of reagent is capable of oxidizing.
Calculation: 5 divided by number of cubic centimeters of urine run in equals per cent. of sugar.
Fermentation Test for Quantity of Sugar in Urine.—If the urine is 70° F. (room) temperature when the specific gravity is taken at both the beginning and end of the test, it will assure accuracy.
To 100 c.c. of urine of known specific gravity, add one-fourth of fresh yeast cake thoroughly broken up. Mix thoroughly and set aside at a temperature between 85° and 95° F. for twenty-four hours, after which time test with Benedict’s or Fehling’s solutions. If reduction is obtained, it will be necessary to allow the fermentation to continue until it is complete. When no further reduction is obtained, the specific gravity is taken after the urine has reached a temperature of 76°. The difference in the specific gravity at the beginning and end of the test multiplied by 0.23 gives the percentage of sugar in the urine.
The following formulas represent the various solutions used in the above test:
Benedict’s Qualitative Solution
| Gm. or c.c. | |
| Copper sulphate (pure crystals) | 17.3 |
| Sodium or potassium citrate | 173.0 |
| Sodium carbonate (anhydrous) | 100.0 |
| Distilled water to make | 1000.0 |
Fehling’s Solution
| (1) Copper Sulphate Solution: |
| 34.65 grams copper sulphate dissolved in water and sufficient water added to make 500 c.c. |
| (2) Alkaline Solution: |
| 125 grams potassium hydroxide. |
| 173 grams Rochelle salts dissolved in water q.s. to make 500 c.c. |
| Keep solution in separate bottles and mix in equal quantities when ready to use. |
Haines’s Solution
| Copper sulphate (pure) | 30 grams |
| (dissolved in ½ oz. (15 c.c.) distilled water) | |
| Add ½ oz. pure glycerin, mix thoroughly, and add 5 oz. liquor potassæ. | |
Benedict’s (Quantitative) Solution
| Copper sulphate (pure crystals) | 18 grams |
| Sodium carbonate (crystallized) (or 100 grams of anhydrous salt) | 200 grams |
| Sodium or potassium citrate | 200 grams |
| Potassium sulphocyanide | 125 grams |
| 5% solution of potassium ferrocyanide | 5 c.c. |
| Distilled water to make total volume of 1000 c.c. | |
Dissolve the carbonate, citrate, and sulphocyanide with the aid of heat and enough water to make 800 c.c. of mixture. (Filter, if necessary.) Weigh exactly the copper sulphate crystals and dissolve in 100 c.c. of water, now add it to the first solution; stirring constantly. Add the ferrocyanide solution; cool and dilute to exactly 1 liter.
50 mg. (0.050 gm.) of sugar will reduce 25 c.c. of the above solution.
Gerhardt’s Ferric Chloride Reaction for Diacetic Acid.—To 10 c.c. of fresh urine, add carefully a few drops at a time of undiluted aqueous solution of ferric chloride U.S.P. A precipitate of ferric phosphates first forms, but upon the addition of a few more drops of the same solution it is dissolved. A Burgundy red (red wine) color is obtained in the presence of diacetic acid. The depth of this color is indicative of the quantity of acid present. Joslin[112] records the intensity of the reaction as follows, +, ++, +++, or ++++.
According to Joslin, it must be remembered that similar reaction is obtained in the urine of individuals taking salicylates, antipyrin, cyanates, or acetates, but it is a simple process to differentiate between the color produced as a result of diacetic acid and that produced by the above-mentioned drugs. If the solution is boiled for two minutes, the color from diacetic acid will disappear, owing to the unstableness of that substance, while that from the drugs will remain unchanged.
Test for Acetone.—Pour 5 c.c. of urine to be tested into a test tube, add a crystal of sodium nitroprusside, acidify with glacial acetic acid, shake well, and then make alkaline with ammonium hydrate. The presence of acetone is indicated by a purple color.