Analytical Sciences

Biomolecule Analyses in an Open-Tubular Capillary Chromatography Using Ternary Mixed Carrier Solvents with Chemiluminescence Detection

Naoya TAKAHASHI, Yuji MASUHARA, Naoya JINNO, Masahiko HASHIMOTO, and Kazuhiko TSUKAGOSHI

Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan

We examined the elution behavior of isoluminol isothiocyanate (ILITC)-labeled biomolecules (α-amino acids, peptides, and proteins) in an open-tubular capillary chromatography system using an untreated fused-silica capillary tube and a water–acetonitrile–ethyl acetate mixture carrier solution. Such an open-tubular capillary chromatography is called “tube radial distribution chromatography (TRDC)” for convenience. A mixture of ILITC and ILITC-labeled biomolecules was analyzed using TRDC with chemiluminescence detection that provided simple instrument without a light source and complex optical devises. The ILITC and the labeled twenty α-amino acids were separated, in this order or the reverse order, or not separated with an organic solvent-rich and water-rich carrier solution. Their elution behavior was considered to be of hydrophilic or hydrophobic nature of ILITC and the labeled α-amino acids. The ILITC and the labeled protein, alcohol dehydrogenase and bovine serum albumin, were separated in this order with an organic solvent-rich carrier solution, while they were eluted in the reverse order with a water-rich carrier solution, based on the TRDC separation performance. The TRDC system worked with the untreated open-tubular capillary tube not using any specific capillary tubes, such as coated, packed, or monolithic.

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