Related: BioStack Compendium for Bits n Bio
In a fluorometer, nucleic acids are quantified using highly sensitive and accurate fluorescent dyes. Separate dyes are used for double-stranded (ds) DNA, single-stranded (ss) DNA or RNA estimation which increases its specificity. Binding of the dye to dsDNA emits fluorescence at a specific wavelength which is then estimated by the fluorometer and the DNA is quantified. This eliminates the possibility of contamination by RNA, free nucleotides and other proteins.
Spectrophotometers use the principle of light absorbance at 260nm by the nucleic acids. In general, it doesn’t distinguish between dsDNA, RNA, proteins or free nucleotides in the sample leading to potentially overestimation of DNA content.