Chromatographia

, Volume 78, Issue 7–8, pp 487–494 | Cite as

Separation Behavior of Short Oligonucleotides by Ion-Pair Reversed-Phase Capillary Liquid Chromatography Using a Silica-Based Monolithic Column Applied to Simple Detection of SNPs

  • Kunio Kawamura
  • Keisuke Ikoma
  • Yoshimi Maruoka
  • Hideaki Hisamoto
Original
First Online:
Received:
Revised:
Accepted:

Abstract

The separation behaviors of short oligonucleotides using capillary liquid chromatography have been investigated comparatively on an octadecylsilane (ODS) packed column (0.3 mm inner diameter, particle size 3 μm) and a monolithic ODS column (0.5 mm inner diameter, through pore size 2 μm). The present paper clearly demonstrates that the resolution obtained using the monolithic ODS packed column is much higher than that using a semi-μ-high-performance liquid chromatography (HPLC) column on a semi-μ-HPLC system. Furthermore, it was surprising that complete baseline resolution of oligonucleotides, typically shorter than 30-mer, with single nucleotide units was achieved using the monolithic column, although this was not possible using the ODS packed column. Good separation of short oligonucleotides was achieved at 40–60 °C with a linear 40–60 % gradient of methanol content. Separation conditions on the monolithic column were optimized regarding the type of counterion, methanol content, temperature, and flow rate. Using the present method, detection of single-nucleotide polymorphisms was achieved without using a mass-spectrometry (MS) detector.

Keywords

Capillary liquid chromatography Ion-pair reversed-phase separation Monolithic column Oligonucleotide Single-nucleotide polymorphism 
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Notes

Acknowledgments

This research was supported by a Grant-in-Aid for Scientific Research 21200004 (2009–2011) on Innovative Areas from MEXT of Japan.

Supplementary material

10337_2015_2855_MOESM1_ESM.pdf (43 kb)
Supplementary material 1 (PDF 43 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Kunio Kawamura
    • 1
  • Keisuke Ikoma
    • 2
  • Yoshimi Maruoka
    • 1
  • Hideaki Hisamoto
    • 2
  1. 1.Department of Human Environmental StudiesHiroshima Shudo UniversityHiroshimaJapan
  2. 2.Department of Applied Chemistry, Graduate School of EngineeringOsaka Prefecture UniversitySakaiJapan

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