Discrete spacers for photometric characterization of humic acids separatedby capillary isotachophoresis

A group of twenty discrete spacers suitable for photometric characterizatio n of humic acids (HAs) after their isotachophoretic (ITP) separation at pH 10 was found. The spacers, inorganic and organic acids and amino acids of s uitable acid-base and migration properties exhibiting no light absorption i n the W region of the light spectrum, made possible to perform this charact erization in a sensitive spike mode of the ITP analysis. Using this approac h a complex mixture of humic constituents present in a test KA 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 tu nes of the spacers. A photometric monitoring of the fractions in the ITP st ack at a 405 nm detection wavelength provided an adequate selectivity and s ensitivity into the characterization. Relative sizes of the detected fracti ons of the test HA preparation ranged from 0.2-0.3 to 27.5% (based on the r esponse of the phototometric detector at 405 nm). The fractions representin g 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 repeatab ility of the total area of the detection signal corresponding to humic cons tituents in the ITP stack was ca. 2.5%. Repeatabilities of the peak areas o f the fractions of the humic constituents defined by the spacers ranged fro m 2 to 6% for the fractions representing 1% or mon of the total area and fr om 8 to 12% for those representing less than 1%. No marks of aggregations o f the humic constituents were detected and reproducible ITP profiles (finge rprints) of the studied humic preparation were achieved under the developed working conditions. (C) 2001 Elsevier Science B.V. All rights reserved.