MICRO-NUTRIENT PROBLEMS 43 



to biological material have been described by Eisler, Rosdahl and 

 Theorell (1936), by Eden and Green (1940), and by Piper (1942). 

 In the procedure of the first group of workers the dried material 

 is heated on a sand-bath with sulphuric acid and perchloric acid 

 until the mixture is colourless or pale yellow. The resulting liquid 

 is cooled under the tap and then rendered slightly alkaline to 

 litmus by the addition of 8-9 per cent ammonium hydroxide. 

 Iron is precipitated by treating the alkaline solution with a few 

 ml. of a 4 per cent solution of sodium pyrophosphate, then 

 heating at 80° C. for 30 min. and cooling to room temperature. 

 If a crystalline precipitate is present at this stage it must be dis- 

 solved by the addition of water. Now 2 ml. of water and 5 ml. of 

 amyl alcohol are added and then immediately 0-5 ml. of a 2 per 

 cent solution of sodium diethyldithiocarbamate. 



The mixture is now strongly shaken and centrifuged. The 

 amyl alcohol layer is separated and its light absorption in light 

 of wave-length 4400 A. measured. The depth of colour of the 

 amyl alcohol layer is dependent not only on the concentration 

 of copper, but also on the salt concentration of the aqueous 

 phase from which the amyl alcohol layer is separated, the salt 

 concentration depending on the amount of sulphuric acid used. 

 Hence it is necessary to use a constant technique in order to 

 obtain reliable results. 



Eden and Green also used a wet ashing method, the material 

 e.g. 5 ml. of blood, 1-5 g. of fresh tissue or 1 g. of dried material, 

 being digested with a mixture of sulphuric and perchloric acids 

 to which nitric acid is added later. After dilution with water the 

 digest is treated with 2 ml. of 50 per cent ammonium citrate 

 and 5 ml. of ammonia (sp. gr. 0-880). The ammonium citrate 

 effects the deionization of the iron and prevents the precipitation 

 of calcium phosphate. The solution is then made up to 25 ml. 

 with water. If the material is relatively low in calcium and 

 phosphorus but high in iron it is preferable to use 10 ml. of 4 per 

 cent hydrated sodium pyrophosphate instead of the ammonium 

 citrate, as the resulting solution is colourless, whereas with 

 citrate the solution is slightly coloured. 



Two ml. of a recently filtered 0-5 per cent solution of sodium 

 diethyldithiocarbamate are now added to the solution, kept 

 constantly shaken, then 5 ml. of amyl alcohol added, and the 



