Preparation of Standard Reagents

Benzidine may be purified by crystallisation from boiling water in which it is soluble to about 1%. When crystallised from water above 80° (i.e., from an aqueous solution heated to boiling, cooled to 80° and filtered through a funnel maintained at 80°), the anhydrous base (M.P. 127°) is obtained in meagre quantity under 80° a hydrate (M.P. 104°—105°) is formed. By crystallisation in the ordinary way, the bulk of the product is the monohydrate. This should be exposed in a desiccator over cone, sulphuric acid until (about 24 hours) a M.P. of 127° is reached. N.B.— The melting points must be taken quickly. (J. S. C. I., 46, 209.) The product obtained by vacuum distillation (see Fig. 22) is also suitable for standardisation purposes. 

To prepare semi-normal nitrite solution, about 37-5 gms. of commercial sodium nitrite (or rather less of a purer salt) are dissolved in water, 

The solution is measured by means of a pipette into a 500-c.c. beaker  

200 gins, of ice and 13 gms. of cone, hydrochloric acid are also added. The beaker is slightly tilted to one side, and the nitrite solution, run in from a burette, is allowed to trickle down the side of the beaker and thus to sink quickly to the bottom. When about 45 c.cs. nitrite have been added, the solution is stirred with a glass rod, and more nitrite is rim in, drop by drop (tests being carried out at intervals), until a drop of the solution just causes an immediate blue coloration on starch-iodide paper.  

After standing a few minutes, a test is again applied to see if the excess of nitrite still remains, and if not, more nitrite is added, until a slight positive test is obtained after a few minutes standing. 

---

This section contains descriptions of basic procedures that occur throughout the book. In this way, we hope to eliminate the repetition of basics such as RNA quantification, gel extraction, extraction with organic solvents, RNA precipitation, long-term storage of RNA, renaturation, the use of spin-columns and preparation of standard reagents. 

The preparation of standards, reagents and culture media must take place according to pertinent specifications care must be taken that they have the required characteristics during the period of use. 

---