Analytical Sciences

Elution Profile of Di-peptides on a Sulfonated Ethylstyrene–Divinylbenzene Copolymer Resin Column by High-performance Liquid Chromatography

Jian GUO,* Tomomi SAIKI,* Kumrungsee THANUTCHAPORN,* Wanying LIU,* Akihiro SHIMURA,** and Toshiro MATSUI*

*Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan
**Separation Materials Laboratories, R&D Center, Kurosaki Plant, Mitsubishi Chemical Corp., 1-1 Kurosakishiroishi, Yahatanishi, Kitakyushu, Fukuoka 806-0004, Japan

This study investigates the characteristics of a partially sulfonated ethylstyrene–divinylbenzene copolymer for the separation of di-peptides by high-performance liquid chromatography. Di-peptides (VE, VA, VH, VK, and VR) with different isoelectric points (pI, 4.0 to 9.7) and log P values (−1.63 to −0.72) were used to optimize the separation conditions of the columns packed with sulfonated copolymer resin. The retention factor (k) of the di-peptides on the column with a 0.81 wt% sulfo group content decreased with increasing concentrations of phosphate salts (2.5 – 20 mmol L⁻¹) in the mobile phase. The complete separation of the five di-peptides was obtained with a gradient of 10% methanol containing 5 mmol L⁻¹ NaH₂PO₄ (pH 4.8) to 50% methanol containing 5 mmol L⁻¹ Na₂HPO₄ (pH 8.9) for 60 min at 0.5 mL min⁻¹ at 50°C. Under the optimal conditions, a good relationship between the k and pI values of the di-peptides, with the exception of VE (pI 4.0), was observed, suggesting that the retention of the di-peptides on the column packed with sulfonated copolymer resin was dependent on the pI value, when it was greater than 5. The log P value also influenced the separation characteristics of the column; peptides possessing the same pI value (6.4 for GH, VH, IH, and FH) showed a higher retention on the column with increasing log P values. In conclusion, the prototype sulfonated ethylstyrene–divinylbenzene copolymer column was applicable for the separation of basic di-peptides, and the separation depended on the pI and hydrophobicity of the di-peptides.

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