246 CU\^TTE COLORIMETRY 



1% Suspension of Norit in 5% Trichloroacetic Acid. First treat 

 the Norit by placing 200 g. in a large flask, add 1 1. 10% hydro- 

 chloric acid, heat to boiling, filter with suction, transfer the cake 

 of Norit to a large beaker and add 1 1. distilled water; stir well, 

 filter, and repeat the whole procedure until the washings give a 

 negative or very faint test for ferric ion. Dry overnight at 110- 

 120°. Some grades of activated carbon may not require washing; 

 this can be determined by running a blank test on trichoroacetic 

 acid washings of the carbon. If these give no more color than the 

 acid alone, the washing of the carbon is unnecessary. Suspend 5 

 g. of the iron-free Norit in 100 ml. 5% trichloroacetic acid. After 

 the Norit has settled, decant the supernatant and restore the 

 volume with 5% trichloroacetic acid. Repeat several times to 

 eliminate some of the very fine floating carbon particles. It is 

 necessary to prevent carbon from getting into the final sample 

 since carbon contamination may result in low values. If difficulty 

 from floating is encountered, add 1 vol. 2% gelatin to 10 vol. of 

 the acid suspension just before use. Once a week or so, replace 

 the supernatant by fresh acid to avoid the possibility of contamin- 

 ation with heavy metals that may slowly leach out of the Norit. 



PROCEDURE 



1. Place 10 /xl. serum in the bottom of a serological tube (6 X 

 50 mm.) ; add 40 jxl. of the acid-charcoal suspension, and mix by 

 tapping the tube. In pipetting the charcoal suspension, first blow 

 through the pipette to suspend the material and then fill and empty 

 it rapidly to prevent the charcoal from settling out. Employ a con- 

 striction pipette with a tip and constriction two to three times wider 

 than normal to avoid plugging. 



2. Cap the tube with a piece of Parafilm or a stopper and centri- 

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



3. Transfer 30 fA. of the supernatant to another serological tube; 

 add 10 fx\. of the osazone reagent, and mix by tapping. 



4. Cap the tube and set aside at 38° for 3 hr. 



5. Chill the tube in ice water and add 50 fil. ice-cold 65% sul- 

 furic acid. Mix very well, and, after 30 min. at room temperature, 

 measure the color intensity at 520 m/x using the 0.05 ml. cuvette with 

 a Beckman spectrophotometer (page 216). If the vol. is increased 

 the 0.2 ml. cuvette may be used. 



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