THE BIOCHEMISTRY OF MILK 147 



can reduce blue cupric (Cu++) to red cuprous 

 (Cu+) ions. It is not given by the sugar most 

 familiar to you, sucrose (cane sugar). Why not? 

 Run the Benedict test with a graded series of 

 sugar concentrations prepared as follows: Label 

 5 small test tubes, #1 to #5. To a pinch of 

 lactose in test tube #1, add 20 drops of water, 

 and swirl to dissolve. Add 10 drops of water to 

 each of the other 4 test tubes. Now transfer 10 

 drops of lactose solution from test tube #1 to 

 #2 and mix; then transfer 10 drops from #2 to 

 #3, and so on. Discard 10 drops of solution 

 from tube #5, after mixing. You now have a 

 series of test tubes, each containing half as much 

 sugar as the one before it. Add 3 ml of Benedict 

 solution to each, and heat in the boiling water 

 bath for 2 minutes. Note the colors, and set 

 aside. 



Completion of the dialysis experiment 



The biuret and Benedict tests can now be used 

 to determine whether protein, sugar, or both 

 have passed out of the dialysis sac prepared last 

 week. Remove the sac from the surrounding 

 solution in the test tube. The latter is called 

 the dialysate. Empty the contents of the sac 

 into a beaker. Test 5 drops of each solution 

 for both sugar and protein. Compare the colors 

 with those of the blanks, prepared above, and 

 with the graded sugar series. Does the dialysate 

 contain sugar? How much sugar, compared 

 with the sac contents? Does it contain protein? 

 How do you explain these observations? 



Paper chromatography of amino acids and 

 casein hydrolysate 



In this experiment, six known amino acids 

 [alanine, aspartic acid, lysine, proline (an imino 

 acid), histidine and methionine], one unknown 

 amino acid, and your casein hydrolysate are 

 chromatographed on a single sheet of filter 

 paper. 



Place a piece of filter paper, 4" X 5", on wax 

 paper, and draw a fine line with lead pencil 

 parallel to and 1.5 cm from one long edge, which 

 will be the bottom of your chromatogram. On 

 this line mark pencil dots about 1 cm apart. 



starting about 2 cm from one edge. These are 

 to indicate the positions for placing your sam- 

 ples; you can label each sample directly on the 

 paper below the line. 



The samples are applied to the paper with a 

 fine glass capillary. (The instructor will show 

 you how to make capillaries.) Draw a little 

 solution into a capillary, touch it to the paper 

 at a pencil dot, let dry, and repeat. Each spot 

 should not be more than 3 mm in diameter. 

 Two such superimposed applications should be 

 sufficient with the amino acid solutions, and 

 four with the casein hydrolysate. It will be 

 advantageous to place your unknown amino 

 acid in the middle, between the third and fourth 

 known amino acid. (Note: Avoid excess han- 

 dling of the filter paper, since your hands might 

 contaminate it with amino acids. Touch it only 

 at the edges.) Now roll the sheet into a cylinder, 

 with the short dimension vertical, and tie the 

 edges together with staples so that they do not 

 touch each other, as shown in the diagram in 

 Exercise IV. 



Pour about 30 ml of solvent (formic acid: 

 isopropanol : water = 10:70:20) into a quart 

 jar. Line the walls of the jar with a piece of 

 filter paper dipping into the solvent, to act as a 

 wick and help to keep the atmosphere in the jar 

 saturated with solvent. Splash the solvent 

 about. Now insert your cylinder, keeping it 

 away from the walls, close the jar, and let it 

 stand quietly. Wait until the solvent has risen 

 within 0.5 cm from the top of the paper before 

 removing the cylinder and letting it dry. Then 

 dip it into the ninhydrin-acetone reagent, and 

 after the acetone has evaporated, place the 

 paper in the warm oven (80°) for a few minutes. 

 Do not leave it too long or let it overheat! Look 

 each minute, and take it out as soon as you can 

 clearly see the spots. Then immediately outline 

 the spots that you see with pencil. (You can 

 now handle the paper freely but the spots fade 

 in the light.) Which of the amino acids is your 

 unknown? 



The ninhydrin test yields purple colors with 

 amino acids and some related substances, and 

 a yellow spot with the imino acid proline. 



