In the Laboratory

A Mini-Qualitative Carbohydrate Analysis Session

Johannes S. Malherbe and Cornelius J. Meyer Department of Chemistry, University of Stellenbosch, Stellenbosch, 7600, South Africa Our nonmajor chemistry students (agriculture, forestry, medicine, etc.) typically follow a one-semester course in organic chemistry, and it is therefore important that they make contact with as many of the relevant functional groups as possible. To ensure that what they learn in the classroom is also translated into a hands-on experience within the limited time available, we make use of flowcharts to streamline some practicals (1). Similarly, we use the following simple but effective carbohydrate session to introduce some visual tests and how they relate to the structural features of the selected carbohydrates. Students have three hours in which to complete this practical. Because they mostly have limited analytical skills, we supply them with a flowchart to ensure that they make efficient use of their time and keep up to schedule (Fig. 1). Experimental Procedure The tests corresponding to the numbers in the flow chart are described below. Methodology for the preparation of reagents is also included. For the benefit of the students the flowchart includes all the carbohydrates used as unknowns with the exception of starch, since starch is the easiest of the carbohydrates in this series to detect. The unknowns are supplied as 1% solutions in water, and each student receives two different numbered samples, one each of a monosaccharide (arabinose/fructose/glucose) and a disaccharide (lactose/sucrose). Although the order of the tests used in the flowchart is not rigid, students are encouraged to use the specified order to ensure a successful and unambiguous identification. The more alert students will soon realize, however, that the only restriction on the order of the tests is to ensure that the Seliwanoff test either immediately precedes the reducing test (e.g., Fehling), or vice versa, to ensure that fructose and sucrose can be distinguished from each other. This flexibility allows the lecturer the freedom of changing the flowchart from year to year. Students are instructed to practice on the known compounds provided, so as to have the benefit of prior knowledge before attempting to identify the unknowns. Misinterpretation of results in general can result when students directly attempt the unknowns, using tests that could be out of sequence when a process of elimination is required. In the discussion leading to the practical, students must be warned that although ketohexoses dehydrate more rapidly than aldohexoses to give furfural derivatives that condense with resorcinol to form a red complex in the Seliwanoff test, prolonged heating of the test solution must be avoided to prevent an aldohexose from giving a false positive test. This also holds true for the Bial test for pentoses, as certain hexoses will also yield color complexes on prolonged heating. Thus, in addition to having practiced beforehand, we suggest that as a further aid in helping them make unambiguous identifications they test references (i.e., a compound that they know will give a negative test and a compound that will give a positive test) parallel

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

Figure 1. Flowchart for identifying unknowns.

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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Journal of Chemical Education Vol. 74 No. 11 November 1997

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.

Test 6: Bials Test for Pentoses

The stock solution for Bials orcinol reagent (2, 3) is prepared by dissolving 1.5 g of orcinol in 500 mL of concentrated HCl, and then adding ca. 30 drops of a 10% FeCl 3 solution. To three separate test tubes each containing 2 mL of Bials reagent add, respectively, 1 mL of arabinose (reference), glucose (or any other negative reference), and unknown. Identify each test tube and heat in the flame until boiling commences. A blue-green color indicates a positive reaction. Compare the color (if any) of the unknown to that of the reference.

Test 3: Tollens Test

The Tollens reagent, Ag(NH3 )2 OH, is prepared by students as follows (1, 4). Add 2 drops of a 5% NaOH solution to 2 mL of a 5% AgNO3 solution in a clean test tube. Add ca. 1 M ammonia solution dropwise until the initial precipitate formed just dissolves. Add 2 mL of the unknown to the Tollens reagent and heat in a water bath at ~60 C for up to 10 min. In a thoroughly clean test tube the silver metal that is formed by reduction of the silverammonia complex is deposited as a mirrorlike coating on the walls of the test tube.

Test 7: Specific Test for Lactose (or Maltose)

To 5 mL of the unknown add 5 drops of a 20% NaOH solution and 5 drops of a 5% methyl ammonium chloride solution (5). Heat the mixture in a flame until a yellow discoloration develops. In a positive test the yellow will gradually be transformed into a deep red. Literature Cited
1. Labuschagne, A. J. H.; Malherbe, J. S.; Meyer, C. J. J. Chem. Educ. 1994, 71, 10881090. 2. Plummer, D. T. An Introduction to Practical Biochemistry, 2nd ed.; McGraw-Hill: London, 1978. 3. Pavia, D. L.; Lampman, G. M.; Kriz, G. S., Jr.; Introduction to Organic Laboratory Techniques a Contemporary Approach, 2nd ed.; Saunders: Philadelphia 1982. 4. Williamson, K. L.; Macroscale and Microscale Organic Experiments; D.C. Heath: Lexington, 1989. 5. Egan, H.; Kirk, R. S.; Sawyer, R. Pearsons Chemical Analysis of Foods, 8th ed.; Churchill Livingstone: London, 1981.

Test 4: Acid Hydrolysis

Mix 1 mL of dilute HCl (3 M) with 5 mL of the unknown carbohydrate and heat the mixture in a test tube in a boiling water bath for 10 min. Neutralize the acidic solution with a dilute NaOH solution (3 M) and test with litmus paper (2, 3). Use 2 mL of this hydrolysate for a Fehlings test as described in Test 2 above.

Test 5: Seliwanoffs Test for Ketohexoses

The stock solution for Seliwanoff s reagent (2, 3) is prepared by dissolving 0.5 g of resorcinol in 1000 mL of HCl (3 M).

Vol. 74 No. 11 November 1997 Journal of Chemical Education

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