Discrete spacers for photometric characterization of humic acids separated by capillary isotachophoresis

A group of twenty discrete spacers suitable for photometric characterization of humic acids (HAs) after their isotachophoretic (ITP) separation at pH 10 was found. The spacers, inorganic and organic acids and amino acids of suitable acid–base and migration properties exhibiting no light absorption in the UV region of the light spectrum, made possible to perform this characterization in a sensitive spike mode of the ITP analysis. Using this approach a complex mixture of humic constituents present in a test HA preparation was separated into 22 fractions migrating in the interzonal boundary layers formed by the zones of discrete spacers and 21 fractions mixed with the zones of the spacers. A photometric monitoring of the fractions in the ITP stack at a 405 nm detection wavelength provided an adequate selectivity and sensitivity into the characterization. Relative sizes of the detected fractions of the test HA preparation ranged from 0.2–0.3 to 27.5% (based on the response of the phototometric detector at 405 nm). The fractions representing ca. 0.2–0.3% of the total peak area could be still quantified when 800 ng of the test preparation was loaded onto the ITP column. A typical repeatability of the total area of the detection signal corresponding to humic constituents in the ITP stack was ca. 2.5%. Repeatabilities of the peak areas of the fractions of the humic constituents defined by the spacers ranged from 2 to 6% for the fractions representing 1% or more of the total area and from 8 to 12% for those representing less than 1%. No marks of aggregations of the humic constituents were detected and reproducible ITP profiles (fingerprints) of the studied humic preparation were achieved under the developed working conditions.