250 CUVETTE COLORIMETRY 



3. Obtain the results from a calibration curve derived by using 

 various amounts of the standard glycogen soln. Subject the glycogen 

 standards to the same steps of initial precipitation and color develop- 

 ment as the unknowns. 



note: Constancy of temperature and iodine concentration are essential, 

 and the glycogen standard should be prepared from glycogen obtained from 

 the same source as the sample to be analyzed. See page 248. 



VITAMIN A AND CAROTENE 



By means of 2 mm. quartz microcuvettes and an adapter for the 

 Beckman spectrophotometer (page 217), which was used for the ab- 

 sorption measurements, Bessey et al. (1946) succeeded in determin- 

 ing the vitamin A and carotene in as little as 35 fA. blood serum. 

 Various volumes of serum greater than 35 /xl. may be used as long as 

 proportional volumes of the reagents are also employed. The pro- 

 cedure to be described is based on the use of 60 ix\. The method in- 

 volves saponification and extraction with solvents of low volatility, 

 measurement of the absorption by the small volumes at 328 m^u ( for 

 vitamin A) and 460 m/^ (for carotene), destruction of the vitamin A 

 absorption by treatment with ultraviolet irradiation, and finally re- 

 measurement of the absorption of 328 m/x. By measurement of the 

 absorption of 328 m/x, before and after the destruction of the vitamin 

 A, absorption at this wavelength due to other substances will not 

 interfere with the determination. 



Method of Bessey et al. for Vitamin A and Carotene 



SPECIAL APPARATUS 



Ultraviolet Apparatus. A diagram of the apparatus used for the 

 ultraviolet irradiation is shown in Figure 81. A mercury discharge 

 lamp (General Electric B-H4) with purple emelope-filter and 

 transformer are used to furnish the radiation. The brightest part 

 of the lamp is placed opposite to the lower half of the tube, and 

 the shadow of the electrode support is not allowed to fall on any 

 tube. The tubes must be cooled during the irradiation by a moder- 

 ate air current from a fan. 



Mixing Apparatus. The device for mixing the liquids in the narrow 

 tubes is made by cutting off the head of an eight-penny nail, 



