The Original Formation of Amino Acids 99 



THE ACTION OF ULTRAVIOLET RAYS 



The irradiation of the solution was carried out in optical cuvettes through 

 laminae of plane-parallel quartz, using a PRK-2 lamp at 25 cm, the intensity 

 of the irradiation being 27 x lo*^ erg/cm^ min during 20 hours. In the main 

 experiments we used aqueous solutions containing 2-5% CH2O and i-o-i'5% 

 NH4NO3 or NH4CI; the volume irradiated was 20 ml. During irradiation the 

 temperature of the solution was about 40-45 °C, in some experiments it was 

 + 1 — h2 °C (the temperature of melting ice). The pH of the solution was 1-5-2, 

 or 5-6-2 when chalk was present. When the irradiation was finished the 

 volatile amines and organic acids were distilled off in a vacuum and the concen- 

 trate (e.g. I ml.) was submitted to chromatography on paper (Leningrad slow) 

 using butanol-acetic acid mixtures as solvents. Chromatography was repeated 

 in most experiments to give better separation of the spots. The chromatograms 

 were coloured with ninhydrin and were then fixed with solutions of copper salt; 

 the fixed spots were of the ordinary rosy colour. Identification of the spots was, 

 however, made more difficult by the possible presence of volatile amides and 

 amines in the concentrate [5]. Preliminary extraction of the concentrate with 

 ether in the modified Soxlilet apparatus for 45 hours only led to a weakening of 

 the spots. We therefore compared the extraction of amines with ether, directly 

 with chromatograms through which butanol and acetic acid had flowed for 77 

 hours (method of repeated chromatography). The dried unsprayed chromato- 

 grams were then extracted with ether for 120 and 168 hours after which they 

 were sprayed. As a result of this we manage to bring about the disappearance of 

 a large spot (Rp ^ — ' 0-4), the identification of which with amines was confirmed 

 with control mixtures of amino acids with diethylamine (hydrochloride). 



To get rid of amides, acid hydrolysis of the eluate of the spots was carried 

 out by refluxing with 15% HCl for 8 hours. The hydrochloric acid was then 

 removed by evaporating six times with water. 



Later we adopted another routine for analysing our experimental mixtures. 

 The separation of the amino acids and organic bases was carried out on an ion- 

 exchange resin, Amberhte anionite (IRA-400) with strongly basic properties. 



Thus, in the beginning, volatile amines were distilled off in vacuo in the 

 presence of barium hydroxide, from the mixtures we obtained, while amides 

 were hydrolysed with 15% HCl for 8-10 hours. 



To separate the amino acids from non-volatile organic bases the mixture was 

 put through a column of anionite at a rate of 9 ml/hr. The bases passed right 

 through this column, while the amino acids were retained in it. The elution of 

 the amino acids was carried out with about 20 volumes of M-HCl which was 

 put through at the same rate as the experimental solution. When the hydro- 

 chloric acid had been removed from the eluate, this was concentrated in vacuo. 

 The completeness of the separation of amino acids from organic bases on the 

 column was checked, using artificial mixtures of amines and amides. The con- 

 centrate of the eluate was further analysed for its component amino acids by 

 paper chromatography using the following solvents : mixtures of butanol, acetic 

 acid and water (4:1: 5), phenol-water and, for the identification of valine. 



