Enigmatic Ion-Exchange Behavior of myo-Inositol Phosphates

The separation of myo-inositol mono-, di-, tri-, tetra-, pentakis-, and hexakisphosphate (InsP₁, InsP₂, InsP₃, InsP₄, InsP₅, InsP₆) was carried out using hydroxide eluent ion chromatography. Acid hydrolysis of InsP₆ (phytate) was used to prepare a distribution of InsPs, ranging from InsP₁ to InsP₅’s and including unhydrolyzed InsP₆. Counting all possible positional isomers (many of which have stereoisomers that will not be separable by conventional ion exchange), 40 chromatographically separable peaks are possible; up to 22 were separated and identified by mass spectrometry. InsPs show unusual ion-exchange behavior in two respects: (a) the retention order is not monotonically related with the charge on the ion and (b) at the same hydroxide eluent concentration, retention is greatly dependent on the eluent metal cation. The retention of InsP₃–InsP₆ was determined to be controlled by steric factors while elution was influenced by eluent cation complexation. These highly phosphorylated InsPs have a much greater affinity for alkali metals (Li⁺ > Na⁺ > K⁺) than quaternary ammonium ions. This difference in cation affinity was exploited to improve separation through the use of a tetramethylammonium hydroxide–sodium hydroxide gradient.