254 DYEING 



equally distributed between the two tanks, and at another (shorter) 

 wave-length when it is all put in one tank. 



Another experiment might be performed thus. Take a flat- 

 sided, wedge-shaped glass tank, and arrange it so that it may be 

 pushed across in a spectrophotometer and the absorption of differ- 

 ent thicknesses of the dye in the tank thus measured. Take two 

 solutions of an orthochromatic dye at different concentrations and 

 find the thickness of the dye solutions that give absorption-curves 

 with the peaks rising to the same height. It will be found that the 

 curves are exactly the same. Now measure the thickness of dye- 

 solution through which the light passed in the two cases. Suppose 

 they stand in the ratio x : i . Then the dye measured at thickness i 

 is x times as concentrated as that measured at thickness x. A 

 substance of which this is true is said to obey Beer's law. Now it is 

 characteristic of metachromatic dyes that they do not obey it. 

 One can find the concentration of any solution of an orthochro- 

 matic dye by use of the spectrophotometer when a single absorp- 

 tion-curve has been obtained with a solution of known concentra- 

 tion, but this is not possible with a metachromatic dye in aqueous 

 solution. 



Increase of temperature acts on metachromatic dyes in the same 

 way as dilution. Toluidine blue at about o-6% is reddish violet at 

 ordinary temperatures, but blue at boiling point. Certain neutral 

 salts (sodium chloride, sodium sulphate, potassium chloride, and 

 especially barium chloride) act like dilution or increase of tem- 

 perature. ^^^ It is remarkable, however, that potassium acetate 

 does not have much effect. ^^^ Increase of acidity acts like increase 

 of temperature, though, as has already been remarked, meta- 

 chromatic effects are sometimes seen even below pH 3. 



Dehydrating agents, such as ethanol and glycerol, are antagonis- 

 tic to metachromasy. Even quite strongly chromotropic sub- 

 stances, such as heparin and agar, show less and less capacity to 

 produce metachromatic effects in increasing strengths of ethanol 

 and lose it entirely when the concentration reaches about 50%; 

 chondroitin sulphate loses it at about 30%.^^'* The chromotropic 

 property manifests itself again, however, when water replaces 

 ethanol. 



Chromotropes are not all alike in their reactions to anti- 

 metachromatic substances. Thus DNA is extremely sensitive to 

 salts but less so to ethanol. ^^* 



At any particular degree of dilution, at any temperature or pH, 



