The reasons for the non-linearity of the standard curve for the calcium-cresolphthalein complexone reaction have been investigated. Calcium forms both 1:1 and 2:1 complexes with cresolphthalein complexone; at low calcium concentrations the 1:1 complex predominates and causes non-linearity. At high calcium concentrations the cresolphthalein complexone concentration becomes limiting, resulting in flattening of the calibration curve. It is recommended that two-point calibration of the assay be performed using a low calcium standard rather than setting on the baseline.
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