QUANTITATIVE CHROMATOGRAPHIC METHODS: PART 5. AN IMPROVED NINHYDRIN–HYDRINDANTIN REAGENT

1961 ◽  
Vol 39 (2) ◽  
pp. 417-425 ◽  
Author(s):  
A. T. Matheson ◽  
E. Tigane ◽  
C. S. Hanes
Keyword(s):  
Amino Acids ◽  
Quantitative Determination ◽  
Filter Paper ◽  
High Stability ◽  
Reagent Blank ◽  
Chromatographic Methods ◽  
Ruhemann’S Purple

An improved ninhydrin–hydrindantin reagent has been developed for the quantitative determination of amino acids and peptides separated on filter paper chromatograms. This represents a modification of a reagent described earlier, which was based in turn upon the well-known solution of Moore and Stein. The present reagent has been in use for several years and has shown the advantages of high stability, extremely low and steady reagent blank values, and approximately stoichiometric yields of Ruhemann's purple for most amino acids. In addition to its use for determinations on excised areas from filter paper chromatograms, conditions are defined for the use of the reagent for determinations of amino acids in solutions.

A Shortened Method for the Fluorometric Determination of Phenylalanine

1969 ◽  
Vol 15 (1) ◽  
pp. 15-23 ◽  
Author(s):  
John A Ambrose
Keyword(s):  
Amino Acids ◽  
Incubation Temperature ◽  
Fluorometric Method ◽  
Fluorometric Determination ◽  
Reagent Blank ◽  
Ninhydrin Reaction ◽  
Fluorescence Reaction ◽  
Fluorescence Level ◽  
Ruhemann’S Purple

Abstract As a result of a study of the classic ninhydrin reaction with amino acids (which results in the production of Ruhemann’s purple), and the fluorescence reaction occurring between ninhydrin and phenylalanine in the presence of a dipeptide, the initial incubation period in the fluorescence reaction was shortened from 2 hr. to 16 min. This improvement in the fluorometric method for the determination of phenylalanine was accomplished by (1) decreasing the initial incubation pH from 5.8 to 5.0, and (2) increasing the incubation temperature from 60° to 85°. A study of the effect of pH on the fluorescence level in both the acid and alkaline reactions demonstrated the presence of fluorescence pH plateaus. These plateaus indicate that the fluorescence level was relatively unaffected by a change in pH within specified ranges. A study of the specificity led to the discovery that the inclusion of a peptide control was necessary to correct for one type of nonspecific fluorescence. To make this correction, it was necessary to add the reading of the serum blank to that of the reagent blank, and to subtract this total from the sample reading.

QUANTITATIVE CHROMATOGRAPHIC METHODS FOR THE STUDY OF ENZYMIC TRANSPEPTIDATION REACTIONS

1955 ◽  
Vol 33 (1) ◽  
pp. 416-427 ◽  
Author(s):  
George E. Connell ◽  
Gordon H. Dixon ◽  
Charles S. Hanes
Keyword(s):  
Amino Acids ◽  
Filter Paper ◽  
Quantitative Estimation ◽  
Chromatographic Methods

A colorimetric ninhydrin method has been applied to the quantitative estimation of amino acids and peptides on paper chromatograms. This method is suitable for the analysis of enzymic transpeptidation reactions. A procedure for the purification of filter paper for chromatography has been developed.

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