Phosphoric acid is a versatile triprotic weak acid in biochemistry, widely used for gentle hydrolysis, precipitation, and phosphate quantification in nucleic acid and lipid analyses.

Chemical Properties

Phosphoric acid (H₃PO₄) features a central phosphorus atom tetrahedrally bonded to one =O and three -OH groups (pKa₁=2.14, pKa₂=7.20, pKa₃=12.37), forming a viscous, hygroscopic liquid (density 1.88 g/mL, melting point 42.35°C). Unlike sulfuric acid, it resists strong dehydration but sequesters divalent cations (Ca²⁺, Mg²⁺) and dimerizes at concentrations above 90% to pyrophosphoric acid (H₄P₂O₇). Laboratory-grade solutions (85–98%) maintain stability across pH 1–13 with low volatility.

Biochemical Applications

In nucleic acid analysis, H₃PO₄ is used to hydrolyze RNA into nucleotides for orcinol-based assays (ribose quantification at 660 nm) or to precipitate DNA in ethanol-containing polysaccharide extracts. In lipid biochemistry, dilute phosphoric acid solutions (0.5–2 M) are employed in Fiske–Subbarow phosphate determination, forming molybdate-blue complexes measurable at 820 nm for organic phosphate detection (e.g., phospholipids). In protein chemistry, it is applied for casein precipitation or histone extraction, while metabolic labeling studies may monitor ³²P incorporation following phosphate tagging.