Solubility in Polar and Nonpolar Solvents: Lipids are insoluble in polar solvents and soluble in nonpolar solvents. For this test, the polar solvent is water; the nonpolar solvent is corn oil:

1. Set up three tubes.
2. Add 1 mL (20 drops) each of the pair of liquids indicated below.
3. Mix the contents of each tube using the vortex genie.
4. Wait 2 minutes.
5. Examine each tube carefully. Has the sample dissolved in the solvent or do you see two separate layers in the tube?
6. Record your observations in Table I below.
7. Save your tubes for the Sudan red test described below.

Sudan Red Test: Sudan red, dissolved in alcohol, is a lipid soluble dye. When Sudan red is added to a mixture of lipids and water, the dye will move into the lipid layer coloring it red:

1. Add 5 drops of Sudan red dye to each tube from the solubility exercise described above, and
2. Mix the contents of each tube using the vortex genie.
3. Wait 2 minutes.
4. Examine each tube carefully. Where is the red color found?
5. Record your observations in Table I below.

Table I. Results of Solubility Test and the Sudan Red Test for Lipids

"Grease Spot" Test: You perform this test every time you buy muffins or doughnuts in a paper bag. Lipids make unglazed paper (brown paper, writing paper) translucent:

1. Put a drop of each sample on a piece of unglazed paper.
2. Draw a circle around the spot with a soft pencil.
3. Write the name of the sample in pencil next to the spot.
4. Allow all spots to dry thoroughly.
5. Hold the paper in front of a light source and observe the spots.
6. Record your observations in Table II below.

Benedict’s Test for reducing sugars: Benedict’s reagent (a blue colored solution containing copper ions) is used to test for the presence of reducing sugars. When a solution containing Benedict’s reagent and a reducing sugar is heated, the copper (II) ions in the Benedict’s reagent are reduced to copper (I) ions and the solution changes from blue to green to orange to red-orange to brick-red. A brick-red precipitate (solid), copper (I) oxide (Cu₂O), may appear in the bottom of the tube. The more reducing sugar present in the mixture, the more precipitate will form.

The half-reaction for Benedict’s Test for Reducing Sugars can be shown as:

2 Cu+2

+

2 e-

2 Cu+1

Each copper (II) ion, Cu⁺², is reduced to a copper (I) ion, Cu ⁺¹, by an electron from the reducing sugar. The reducing sugar is oxidized as a result of giving up its electron.

How to Proceed to Test for Reducing Sugars

Note: All groups will set up tubes 1-6 in Table III below. Groups 1,3, and 5 should also set up tubes 7-10, for a total of ten tubes. Groups 2, 4, and 6 should also set up tubes 11-13 for a total of nine tubes. Everyone is responsible for recording and knowing all results, so you should share information.

1. Set up tubes 1-6 and either 7-10 or 11-13, depending on group number. Label each tube with a wax pencil.
2. Add 2 mL (40 drops or one full dropper) of the sample to be tested.
3. Add 2 mL of Benedict’s reagent. What color is the reagent?
4. Mix the contents of each tube using the vortex genie.
5. Record the color of the contents of each tube in the table as soon as you mix them.
6. Place all the tubes in a beaker of boiling water for 1 or 2 minutes.
7. Remove the tubes and allow to cool for 1 or 2 minutes.
8. Observe each tube carefully. Record in Table III the color of any precipitate formed (it may take a few minutes for the precipitate to be visible at the bottom of the tube). Place a star next to the tube with the most precipitate.

Lugol’s Test (Iodine solution) for Starch: Lugol’s reagent contains iodine and potassium iodide (I₂KI). It changes from a yellowish-brown to blue-black in the presence of starch.

How to Proceed to Test for Starch

1. Set up a porcelain spot plate; number each depression in the plate with a wax pencil.
2. Add 3 drops of each sample to be tested to a different well in the spot plate, and make a list of what sample corresponds to what number.
3. Stir with a toothpick.
4. Add 1 drop of Lugol’s reagent to each sample.
5. Record the final color of the contents of each well in Table IV.

Biuret Test for Proteins: Biuret reagent is a light blue solution which turns purple when mixed with a solution containing protein. When the copper ions of the Biuret Reagent react with peptide bonds in the polypeptide chains, a purple color complex is formed.

Note: All groups will set up tubes 1-4 in Table V below. Groups 1, 3, and 5 should also set up tubes 5-8 for a total of eight tubes. Groups 2, 4, and 6 should also set up tubes 9-13 for a total of 9 tubes. Everyone is responsible for recording and knowing all results, so you should share information.

1. Label a set of tubes (see note above) with a wax pencil.
2. Add 2 mL (40 drops) of sample to each tube.
3. Add 2 mL of biuret reagent to each tube. What color is the biuret reagent?
4. Mix the contents of each tube using the vortex genie.
5. Wait 2 minutes.
6. Examine each tube carefully. Note the color.
7. Record your observations in the Table V.

Ninhydrin Test for Amino Acids: Because amino acids contain a free amino group, they are readily detected with ninhydrin reagent which reacts with free amino groups to form a purple or violet colored substance. If a protein is first heated or digested to hydrolyze the protein into free amino acids, the ninhydrin reagent will detect the amino acids.

1. Put a drop of each sample—standards and foods—on a piece of filter paper (you may need a second piece of filter paper).
2. Draw a circle around the spot with a soft pencil.
3. Write in pencil the name of the sample next to the spot.
4. Allow all spots to dry thoroughly.
5. Your instructor will ask the laboratory technician to spray the paper in a fume hood (ninhydrin is toxic).
6. Wait 20 minutes. The completed ninhydrin test papers will be returned to the lab so that you may record the results of the test.
7. Observe each spot carefully.
8. Record your observations in the table.
9. Look up the molecular structure of the amino acid proline. Do you notice something odd about its amino group?