252 CUVETTE COLORIMETRY 



Kerosene-Xylol. Mix equal vol. xylol, C. P., and water white odor- 

 less kerosene (obtainable from Einier and A7nend). 

 Anhydrous Propionic Acid. 



PROCEDURE 



1. Place 60 fxl. serum and 60 [A. alcoholic potassium hydroxide 

 in a test tube 100 X 3 mm. (Prepare the tubes by cutting 200 mm. 

 lengths of glass tubing, 3-3.5 mm. internal diameter, cleaning with 

 boiling half -cone, nitric acid, rinsing, drying, and dividing in the 

 middle with a blast lamp flame to yield two tubes.) If the liquids do 

 not run to the bottom, send them down with a whipping motion. 



2. Mix the liquids, immerse the tube in a 60° water bath for 20 

 min., cool, and add 60 /A. kerosene-xylol. 



3. Extract by holding the tube at a 45° angle against the whirl- 

 ing nail so that the contents are violently agitated for 10-15 sec. 

 Then, when the tube is at room temperature or a little below, centri- 

 fuge for 10 min. at 3000 R.P.M. 



4. Cut the tube with a file just above the kerosene-xylol layer 

 and pipette this layer into the cuvette with a constriction pipette, 

 taking care to avoid the inclusion of any of the aqueous liquid which 

 would cause turbidity. (Use an uncalibrated 50-60 fA. constriction 

 pipette (page 172) with a fine tip and a fine constriction because of 

 the low surface tension of the organic solvents.) 



5. Read the absorption at 460 and 328 m/x, and then transfer the 

 liquid to a soft glass test tube 40 mm. long and 2.5-3.0 mm. internal 

 diameter. 



6. Irradiate with ultraviolet for 30-60 min. (Find the proper 

 time by testing with known vitamin A solns. The time should be six 

 to eight times that required to destroy half the vitamin in pure soln. ) 



7. Transfer the liquid back into a cuvette and take a second 

 reading at 328 m/A. Rinse the pipette with anhydrous propionic acid 

 before the transfer to eliminate traces of moisture which would cause 

 turbidity. 



CALCULATION 



^460 X 480 = microgram per cent carotene 

 (^328 — -2^328 after irradiation) X 637 := microgram per cent 



vitamin A 



