Low-molecular-mass isoelectric point (pI) markers are a reasonable alternative to commonly used peptide and protein pI markers in the field of isoelectric focusing. We present a comparative study dealing with characterization of 44 nitrophenol-based compounds synthesized by the Mannich reaction which pI values cover a pH range from 3.2 to 10.4. The values of pIs of all the presented nitrophenol-based compounds were precisely determined by capillary isoelectric focusing technique (cIEF) when a standard deviation of measurement reached a value of a few hundredths of a pH unit (n 3). Differences have been found between in silico analysis of acid-base properties of the investigated compounds and the experimental cIEF data especially at the basic pH region. The data from the acidic pH range showed better correlation between these methods. Only three compounds were excluded from the group of pI markers because they contained impurities originating from the synthesis or long-term storage. Based on the presented results we identified 41 nitrophenol-based pI markers (NPIMs) which can fulfill requirements of the most applications in a field of isoelectric focusing analyses.

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