Spectrophotometry of nucleic acids by their effect on the complex of cobalt(II) with 4-[(5-chloro-2-pyridyl)azo]-1,3-diaminobenzene

A new ternary azo-dye complex reaction involving nucleic acids has been studied to provide a sensitive spectrophotometric method for nucleic acids. In alkaline conditions, single-stranded nucleic acids react with the complex of Co(II) with 4-[(5-chloro-2-pyridyl)azo]-1,3-diaminobenzene (5-Cl-PADAB), resulting in purple ternary compounds with maximum absorbance at 545.0 nm against the binary complex blank. The mole ratio of Co(II) to 5-Cl-PADAB in the ternary compounds is 1:2, and each nucleotide residue can bind two binary complex molecules. There are linear relationships between the absorbance and the concentrations of the tested nucleic acids, such as calf thymus DNA, fish sperm DNA and yeast RNA in the range 0–4.0 μg ml−1. The 3σ limit of detection for calf thymus DNA is 40 ng ml−1, the relative standard deviation at 3.0 μg ml−1 is 0.79% (n = 10) and the Sandellʹs sensitivity is 5.3 ng cm−2. The same parameters for fish sperm DNA are 41 ng ml−1, 0.84% and 5.4 ng cm−2, and for yeast RNA are 49 ng ml−1, 0.58% and 6.2 ng cm−2. Synthetic samples were analyzed satisfactorily.