Application of gradient programs for the determination of underivatized amino acids and small peptides in reversed-phase high-performance liquid chromatography with contactless conductivity detection

The application of gradient methods in HPLC determinations with capacitively coupled contactless conductivity detection was successfully demonstrated for non-UV-absorbing amino acids as well as for small peptides as analytes. The drift in baseline which is caused by the change in the conductivity of the mobile phase during the elution could be minimized by careful optimization of the composition of the mobile phase. For such conditions the remaining baseline drift could be corrected for either by subtraction of a blank chromatogram or by baseline normalization using a software package for chromatographic data processing. Reverse phase separations were carried out using aqueous eluents containing either methanol or acetonitrile and acetic acid or trifluoroacetic acid. Detection limits between 1 and 40 μM were achieved for the amino acids and the di- and tri-peptides investigated. The reproducibilities for the peak areas were typically found to be about 2% and good linearity was obtained for the concentration range tested (up to 1000 μM).