Abstract
Methods for the estimation of guanidine and its methyl derivatives have been subject to considerable criticism. A study of the color reaction for guanidines recently described by Marston 1 was undertaken in order to determine its biochemical applicability. Although Marston specifically states that his reagent will keep indefinitely, in our hands deterioration and precipitation invariably set in shortly after mixing the three ingredients, sodium nitroprusside, potassium ferrocyanide and sodium hydroxide.
A reagent has been developed, which, although it contains the same ingredients as employed by Marston, has the advantage that it keeps well and gives within five minutes a full color development which does not fade or become turbid for more than an hour, thus allowing ample time for color comparison.
The reagent we now employ is prepared as follows: 6 gm. sodium nitroprusside and 8.5 gm. potassium ferrocyanide are dissolved in water and made up to 100 cc. About 15 to 20 minutes before using, one volume of this solution is mixed with one volume of 10 per cent sodium hydroxide and two volumes of 3 per cent hydrogen peroxide. It has ken found convenient to add 1 cc. of the prepared reagent to 4 cc. of the unknown guanidine solution and compare in a colorimeter with standards similarly prepared. With this technique quantities of guanidine bases as small as 0.2 mg. may be estimated.
Creatine and creatinine give a faint coloration with the reagent but do not interfere materially in the estimations. Uric acid and ammonia interfere by hindering the color devlelopment with the bases. Urea produces about one tenth of the color given by guanidine, a fact not mentioned by Marston. Methyl urea, β-methyl hydantoin, β-methyl liydantoic acid and glucose yield no color, while ethyl alcohol gives a very faint reaction.
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