A convenient microtitration method for the estimation of amino acids.

This paper presents a convenient micromethod for the estimation of amino acids, based on the reaction with ninhydrin. The ammonia formed in this reaction is determined, after elimination of the interference from ninhydrin with superoxol, by addition of saturated potassium hydroxide, aeration, and titration with standard acid. The application of the reaction with ninhydrin is one of the most specific methods for the estimation of amino acids, as the presence of an amino group in the CY position to a carboxyl group is required. Van Slyke and his coworkers have studied this reaction for the determination of amino acids. They pointed out that with most amino acids carbon dioxide, ammonia, and aldehydes, among other products, are evolved, proline and hydroxyproline being exceptions. Thus, it should be possible to estimate amino acids by measurement of the carbon dioxide, ammonia, or aldehydes. They have developed the method by measurement of the carbon dioxide evolved, either manometrically or titrimetrically, the manometric method being preferred (14). MacFadyen (5) published a method based on the ninhydrin reaction, in which the ammonia formed is measured manometrically or titrimetrically. He stated that special precautions must be taken in order to obtain quantitative results, and, even so, low values are obtained with certain amino acids. The ammonia, if allowed to react with the ninhydrin above pH 4.0, forms a stable blue complex and cannot be quantitatively recovered. MacFadyen prevented this by carrying out the reaction at pH 2.5 or lower, and then, before adding alkali, removed the ninhydrin by precipitation with hydrogen sulfide as hydrindantin. Quantitative results were not obtained with some amino acids, notably glycine, a.lanine, tryptophane, proline, and hydroxyproline, indicating that the ammonia may also be bound by certain breakdown products of some amino acids besides ninhydrin itself. We had been working on this problem apparently simultaneously with MacFadyen. Similar difficulties were experienced, but the method of solving them is quite different. The entire determination is carried out in one vessel, and there are no quantitative transfers except for the aeration.