230 HAEMOGLOBIN. 
1. By the action of reagents exerting a reducing action. — 
It is an essential condition which all reagents to be employed in the 
reduction of oxyhemoglobin to haemoglobin must fulfil, that they do not 
act destructively on these substances, as is the case with acids and salts 
possessed of an acid reaction. 
Ordinary solutions of ferrous sulphate or stannous chloride cannot, 
for instance, be employed, as they instantly lead to a decomposition 
of the blood-colouring matter. The first and still the most generally 
employed reducing agents, the use of which dates back to the researches 
of Stokes on the blood-colouring matter, are ferrous and stannous salts 
and the alkaline sulphides. Utilising the well-known property of citric 
and tartaric acids to prevent the precipitation of the salts of iron and 
tin by ammonia and the alkaline hydrates, Stokes indicated easy 
methods of preparing active solutions of ferrous and stannous salts for 
the study of the reduction of oxyhemoglobin. 
(a) Alkaline solutions of ferrous salts (Stokes' reagent). — To a solution 
of a ferrous salt (usually ferrous sulphate or ferrous ammonium sulphate) 
(Fe(]S[H ; ) 2 (S0 4 ) 2 .6H 2 1 ), citric or tartaric acids or one of their alkaline 
salts is added, and then ammonia, until the reaction is alkaline. A light 
green solution is thus obtained, which rapidly darkens in the presence of 
air by the absorption of atmospheric oxygen. Such a solution, which 
must be freshly prepared, exerts a powerful and exceedingly rapid 
reducing action on oxyhemoglobin, even in the cold, Alkaline ferrous 
solutions possess the disadvantage, in proportion as they absorb oxygen 
and become oxidised, of becoming coloured, and absorbing the more 
refrangible rays of the spectrum, interfering, therefore, with the accurate 
study of the specific absorption due to the colouring matter. 
(b) Alkaline solutions of stannous salts. — These are made as described 
under a, by substituting a stannous (usually SnCl 2 ) for a ferrous salt. 
As they do not become coloured on salt being oxidised, these solutions 
do not interfere with the accurate study of the absorption of the violet 
rays. Like the analogous ferrous solutions, those containing tin rapidly 
reduce hemoglobin even in the cold, 
(c) Solutions of the alkaline sidphides. — Solutions of these salts 
(ammonium sulphide being almost invariably employed) effect the 
reduction of oxyhemoglobin, but much more slowly than is the case 
with a and b, and their action is greatly accelerated by heat. Solutions 
of ammonium sulphide for this purpose should be freshly prepared, and 
be protected from the action of atmospheric oxygen and light, which 
bring about chemical changes, and cause them to assume a yellow colour 
and to absorb the violet end of the solar spectrum. 
Solutions of the crystalline sodium monosulphide (Na 2 S) cannot be 
employed with advantage as reducing agents for oxyhemoglobin, as, according 
to my experiments, they lead at once to the formation of sulphomethsemo- 
globin, so that the pure spectrum of reduced haemoglobin cannot be observed. 
(/) Agitation with finely -divided iron^ or with metallic iron reduced 
by hydrogen — the so-called officinal fcrrum rcdactum. 2 
1 Rollett, " Versuche ueber thatsachliche \md vermeintliclie Beziehungen d. Blutsauer- 
stoffes," Sitzungsb. d. I: Alv.d, d, JF-isseJisch., Wien, 1866, Bd. lii. Abth. 2, S. 246 et seq. 
2 Ludwig und Schmidt, "Das Verhalten der Gase welche mit dem Blut durch den 
reizbaren Saugethiermuskel stromen," Sitzungsb. d. k. Sachs. Gesellsch., Leipzig, 1868, Bd. 
xx. S. 12-72. 
