NITRO COMPOUNDS 103 



lution: under these conditions the reaction with benzoyl chloride 

 is patchy, and also the sodium hydroxide extracts much material 

 from the sections. I have found that a much more reliable tech- 

 nique is to use a 5 percent solution of benzoyl chloride in dry 

 pyridine, for about 12 hours. 



There is also some doubt as to whether nucleic acid can, in fact, 

 react with diazonium hydroxides under the circumstances sug- 

 gested by Mitchell. It is certainly true, however, that after ben- 

 zoylation, treatment with a diazonium hyroxide produces a dis- 

 tribution of colour which is very similar to the distribution of 

 nucleic acid revealed by other processes. Further inquiry into 

 the chemistry of this reaction is clearly necessary. 



Nitro Compounds as Chromogenic Reagents 



There are many nitro compounds in use in organic chemistry 

 as analytical reagents. Many of these will react with nucleic 

 acids and proteins. Some of these substances give a reaction 

 product which is coloured. Thus, according to Sanger, dinitro- 

 fluorobenzene will react with tyrosine, histidine, NH 2 groups, 

 and SH groups. Of these, the reaction products with histidine, 

 NH 2 groups, and SH groups are coloured. Usually, however, the 

 intensity of colour is too low to be satisfactory for cytochemical 

 problems. The intensity of the colour may be increased to a sat- 

 isfactory degree by reduction of the nitro group of the reagent 

 to an amino group, diazotisation of the resulting amino group, 

 followed by linkage to a phenol or amine. This procedure pro- 

 duces an azo dye, which is linked by covalent bonds to the 

 group which is being studied. A typical reaction is the following: 



N0 9 N0 2 



Pr-NH 2 -> Pr-NH<^ >N0 2 -> Pr-NH<" ^NH 2 



i 

 N0 2 

 <- Pr-NH/~~^)>N=N-OH 

 N0 2 

 Pr-NH<^ ^>N=N<^ "\NH 9 



