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Arabinose/Fructose/Glucose/Lactose/Starch/Sucrose
I2 (1) (+) Starch (-)
Arabinose/Fructose/Glucose/Lactose/Sucrose
Fehling (2) or Tollens (3) (-) Sucrose
(+)
Arabinose/Fructose/Glucose/Lactose
Seliwanoff (5) (+) Fructose (-)
Confirm with acid hydrolysis (4) followed by Fehling (2) / Tollens (3)
Arabinose/Glucose/Lactose
Bial (6)
(+) Arabinose
(-)
Glucose/Lactose
Methyl amine test (7)
(+) Lactose
(-) Glucose
with their unknowns. This helps to avoid any confusion concerning the actual visual appearance (color) of a positive test, particularly in the case of the Seliwanoff and Bial tests.
Test 1: Iodine Test The iodine stock solution (2, 3) is prepared as follows. Dissolve 2 g of KI in 50 mL of water, add 1 g of iodine, and shake until dissolved. Dilute this solution to 100 mL with water. Add a few drops of iodine solution to 2 mL of the unknown. A dark blue color is a positive test for starch in this series. If the color is very intense, diluting with water will yield a recognizable blue. The students are also encouraged to demonstrate the lability of the color complex, which is due to iodine molecules caught in the helix form of the -D -glucopyranose chains, by heating the contents of the test tube in a water bath and cooling thereafter.
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In the Laboratory
Test 2: Fehlings Solution
The following two stock solutions (4) are required for this test. Solution A: dissolve 34.64 g of CuSO4 5H2 O in 500 mL of water. Solution B: dissolve 173 g of sodium potassium tartrate (Rochelle salt) and 65 g of sodium hydroxide in 500 mL of water. Mix 1 mL of solution A and 1 mL of solution B in a test tube and add 2 mL of the unknown carbohydrate to this mixture. Heat in a flame to boiling point. In a positive test, a muddy green suspension is obtained that on settling yields a red deposit of Cu2O. If preferred and available, the more sensitive reagent 2,3,5-triphenyl-2H-tetrazolium chloride (commonly referred to as Red Tetrazolium) (4) can be used instead of Fehling. To three separate test tubes each containing 2 mL of Seliwanoff s reagent add, respectively, 2 drops of fructose, a negative reference (e.g., glucose), and unknown. Identify each individual test tube and place them simultaneously in a boiling water bath. Heat for approximately 68 min, or until the reference (fructose) reacts to produce a deep red solution. Compare the time the unknown solution takes to acquire a red color (if any), as well as the intensity of the color, to that of the reference.
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