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Dihydrogenphosphate, ${\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4^{-}$, is the singly charged anion obtained when phosphoric acid loses one proton. It is one protonation state of orthophosphate: one phosphorus atom remains connected to four oxygen atoms. Dihydrogenphosphate is the conjugate base of phosphoric acid and the conjugate acid of hydrogenphosphate.^[ref]

For example, reaction with hydroxide can transfer a proton from dihydrogenphosphate to the added base:The total electric charge is $-2$ on either side, and the number of phosphorus atoms is unchanged. This is an acid-base reaction, rather than the breaking of a pyrophosphate bridge. Together, dihydrogenphosphate and hydrogenphosphate can form a buffer solution, accepting added base or acid through interconversion. Their relative amounts at chemical equilibrium depend on pH and the relevant dissociation constant; a total phosphate measurement alone does not determine the amount of dihydrogenphosphate.

Hydrogenphosphate, $\mathrm{HP}{\mathrm{O}}_4^{2-}$, is the doubly charged anion produced by removing a proton from dihydrogenphosphate. It is therefore the conjugate base of dihydrogenphosphate, and it is also the conjugate acid of fully deprotonated orthophosphate, $\mathrm{P}{\mathrm{O}}_4^{3-}$. These species share the same single-phosphorus, four-oxygen framework.^[ref]

The remaining proton allows hydrogenphosphate to participate on either side of an acid-base reaction:In the first reaction, hydrogenphosphate accepts a proton; in the second, it donates one. For a simple accounting example, one mole of $\mathrm{N}{\mathrm{a}}_2\mathrm{HP}{\mathrm{O}}_4$ supplies one mole of phosphate units and two moles of sodium counterions when dissolved. Subsequent proton transfers can change how those phosphate units are distributed between chemical species without changing the total amount of phosphorus. A stated concentration of total inorganic phosphate must therefore be distinguished from the concentration of hydrogenphosphate itself.

An inorganic pyrophosphatase is an enzyme that catalyses hydrolysis of free inorganic pyrophosphate into two orthophosphate units. One explicitly balanced choice of ionic forms isActual protonation and metal binding depend on pH and solution conditions. Hydrolysis removes pyrophosphate generated by other reactions, helping couple those reactions to product removal.

In 1952, M. Kunitz isolated crystalline inorganic pyrophosphatase from baker's yeast. The preparation hydrolysed free pyrophosphate, required activating metal ions such as magnesium, and did not interchangeably hydrolyse the tested organic diphosphate-containing compounds. Purifying a catalyst and comparing substrates connected the reaction to a specific protein activity.^[ref]

For a stoichiometric assay example, complete hydrolysis of $10\mu \mathrm{M}$ pyrophosphate adds $20\mu \mathrm{M}$ orthophosphate, provided no other reaction consumes or generates it. Pre-existing phosphate must be measured as background. Stoichiometry predicts product amount; enzyme kinetics, pH and metal availability determine the time required. The yeast preparation's preferred conditions should not be assigned to every inorganic pyrophosphatase.

Orthophosphate denotes the uncondensed, single-phosphorus phosphate unit. Its fully deprotonated anion is $\mathrm{P}{\mathrm{O}}_4^{3-}$: one phosphorus surrounded by four oxygen atoms. In aqueous biochemical usage, inorganic phosphate, ${\mathrm{P}}_{\mathrm{i}}$, can include protonated forms such as hydrogenphosphate, $\mathrm{HP}{\mathrm{O}}_4^{2-}$, and dihydrogenphosphate, ${\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4^{-}$. Their proportions depend on pH; the name alone does not specify a unique protonation state.^[ref]

The contrast with pyrophosphate is connectivity. Pyrophosphate joins two phosphorus centres through an oxygen bridge; hydrolysis breaks that bridge to yield two separate orthophosphate units. One charge-balanced representation isThe exact proton balance changes with the chosen chemical species, but two phosphorus atoms remain two phosphorus atoms. M. Kunitz's 1952 isolation of inorganic pyrophosphatase from Saccharomyces cerevisiae provided a purified enzyme that specifically catalysed conversion of inorganic pyrophosphate to orthophosphate, making this distinction experimentally measurable.^[ref]