HPLC separation of oligonucleotides in isocratic and temperature-programming mode
N M Djordjevic1, F Houdiere, P Fowler
1Pharma Discovery, Novartis Pharma Inc., Building 503/1106, Basle CH-4002, Switzerland.
Analytical Chemistry
|June 10, 2011
Summary
Isocratic separation rapidly resolves short nucleotide polymers in HPLC. Temperature programming significantly reduces analysis time for longer oligonucleotides, enhancing efficiency for mass spectrometry applications.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Chromatography
Background:
- High-performance liquid chromatography (HPLC) is crucial for analyzing nucleotide polymers.
- Optimizing separation conditions is essential for efficient analysis of diverse oligonucleotide lengths.
Purpose of the Study:
- To investigate isocratic separation and temperature programming for resolving nucleotide polymer mixtures using HPLC.
- To determine the impact of mobile phase composition and temperature on separation efficiency and analysis time.
Main Methods:
- Isocratic HPLC separation was employed for short oligonucleotides (pd(A)(12-18)).
- Temperature programming during HPLC was utilized for separating longer oligonucleotides (pd(A)(25-30) and pd(A)(40-60)).
- Analysis was performed using low concentrations of ion-pairing agents compatible with mass spectrometry.
Main Results:
- Isocratic separation achieved resolution of short oligonucleotides in under 4 minutes.
- Analysis time was highly sensitive to organic modifier concentration in the mobile phase.
- Temperature programming reduced analysis time by 75% for longer oligonucleotides compared to isocratic/isothermal runs.
Conclusions:
- Both isocratic separation and temperature programming are effective HPLC strategies for nucleotide polymer analysis.
- Optimized methods offer rapid and efficient separation suitable for downstream mass spectrometry.
- Low ion-pairing agent concentrations ensure compatibility with electrospray ionization mass spectrometry.
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