452 



J. A. BASSHAM, M. KIRK 



TABLE III 



CALCULATION OF ^^C + '*C FOR STEADY STATE EXPT. li 



Volume 



%co. 



fitnotes 



mC"C 



Specific activity 



A Gas phase at start 

 B "CO2 loop 

 C Dissolved CO^, HCO3 

 D NaH^COj injected 



Total 

 C + D 



895 

 125" 



T092 



1.6 



585 



156 

 81.6 

 40.8 



863 



122.4 



o 



3767 



607.5 



4375 

 607.5 



5.07 /iiClfimole 

 4.95 fiClfimo\e 



' Effective volume. 



warmed at 60° for 10 min. After centrifugation and a further extraction with i ml of 

 water, the combined clear extracts are concentrated at reduced pressure at below 

 room temperature. The concentrated extract, or an aliquot portion thereof, is trans- 

 ferred quantitatively to the paper chromatogram and analyzed in two dimensions 

 (phenol-water, butanol-propionic acid-water) as in earlier work^. The location of 

 the radioactive compounds on the chromatograph is found by radioautography 

 with X-ray film. When necessary, overlapping phosphate esters are eluted, treated 

 with phosphatase and rechromatographed. 



Determination of radioactivity in compounds 



The amounts of radiocarbon in each compound of interest on the chromatograms 

 from each sample is measured with a Geiger-Mueller tube. The paper chromatogram 

 is placed on top of the radioautograph, which rests on a horizontal light table, so 

 that the darkened areas of the film may be seen through the paper. The Geiger- 

 Mueller tube has a Mylar window, gold-sputtered for conductivity, but transparent 

 and thin (less than i mg/cm^) to permit the passage of "C beta particles. This tube 

 has an effective counting area of uniform sensitivity of about 17 cm^. The top of the 

 tube is transparent plastic so that paper and radioautograph may be viewed through 

 the top of the tube. Thus the counting area of the tube may readily be placed in posi- 

 tion over the radioactive compound on the paper. If the radioactive area is more than 

 4 cm across, or if it contains more than 20,000 counts/min (as counted by this tube 

 on the paper), the radioactive area is divided into smaller areas which are counted 

 one at a time (with the remainder of the spot covered by cards). The counting gas 

 used is helium-isobutane (99: i). The counting voltage is about 1300 V. The sensitivity 

 of the counter for '^C beta particles in an infinitely thin layer on an aluminum planchet 

 is about one count/3.1 disintegrations. However, only about one-third of the beta 

 particles escape from the paper (Whatman No. 4) and the actual sensitivity of this 

 tube for ^*C in compounds on the paper is about 1/11.2. These sensitivities were 

 determined by comparison of counts from three aliquot portions of a known ^■'C 

 labeled solution : (a) chromatographed on paper, (b) dried on a planchet, and (c) placed 

 in a scintillation counter with an internal standard. The radioactivity of each com- 

 pound is counted on each side of the paper and an average is taken of the counts 

 from the two sides. Comparison with determinations of radioactivity of compounds 

 quantitatively eluted and placed on planchets indicates that this method of counting 

 gives an accuracy of ± 5 %• 



108 



