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Harnessing HILIC separations for Oligonucleotide Analysis: A Comprehensive Guide and Practical Tips

In the context of oligonucleotide analysis, it can be challenging to distinguish shorter sequences resulting from coupling failure from the desired product, especially with longer oligos. Hydrophilic interaction liquid chromatography (HILIC) is a powerful technique for this purpose. Here, we will discuss the principles of HILIC, its applications in oligonucleotide analysis, and its advantages over traditional ion-pairing HPLC (IP-HPLC). HILIC separates compounds based on hydrophilicity, a form of normal phase chromatography, unlike IP-HPLC which uses a reverse phase system. The more polar stationary phase in HILIC compared to the mobile phase allows for the retention and separation of hydrophilic compounds. Oligonucleotides, being inherently hydrophilic molecules due to their negatively charged phosphate backbone and polar nucleobases, are well-suited for analysis using HILIC.

A vial containing DNA to be purified by HILIC

Key Components of HILIC for Oligonucleotide Analysis:

  1. Stationary Phase: HILIC columns typically use polar stationary phases such as hydroxylated silica, amino-bonded silica, or amide-bonded silica. These stationary phases interact with the hydrophilic oligonucleotides through hydrogen bonding and electrostatic interactions, enabling their retention and separation.

  2. Mobile Phase: The mobile phase in HILIC is typically an organic solvent-water mixture with a high percentage of organic solvent. The composition of the mobile phase can be optimized to achieve the desired separation of oligonucleotides based on their hydrophilicity. Typically, acetonitrile is used as the organic solvent, given its general tendency to result in sharper chromatographic peaks. Oftentimes, a buffering additive is included to improve chromatographic resolution and help with ionization if the system is coupled to a mass spectrometer.

  3. Gradient Elution: In HILIC, gradient elution enhances oligonucleotide separation based on hydrophilicity differences. Altering mobile phase composition throughout chromatography enables distinct elution times for various oligonucleotide species. Given HILIC's normal phase nature, injecting analytes in an organic solvent-water mix is crucial to prevent early retention time interference from excessive water injection. Typically, shorter oligonucleotides will elute earlier than longer ones, due to their lower number of negative charges.

Applications of HILIC in Oligonucleotide science:

  1. Sequence Analysis and purity assessment: HILIC can be used to separate and analyze oligonucleotides with different sequences, allowing for the identification of sequence variants and modifications. HILIC is also very effective at separating nucleotides of different lengths, making it an ideal tool for detecting shortmers and other synthetic impurities.

  2. Structural Analysis: HILIC can offer insights into the secondary structure of oligonucleotides by separating various conformations or modifications. This is particularly evident when the analysis is conducted at a reduced column temperature, leading to decreased conformational shuffling.

  3. Quantitative Analysis: HILIC can be used for the quantitative analysis of oligonucleotides in complex samples, such as biological extracts or pharmaceutical formulations.

  4. Liquid chromatography-mass spectrometry (LCMS) analysis of oligonucleotides: Additives in HILIC (typically ammonium acetate) are volatile and do not hinder ionization, making HILIC a growing separation method for mass spectrometry coupling. IP-HPLC, the predominant separation technique in this context, suffers from the drawback of using ion pairing agents that can greatly suppress MS signals. Although some pairing agents have been created to address this issue, they present various challenges, including toxicity, high cost, and limited shelf life.

HILIC is a robust chromatographic method for analyzing oligonucleotides, providing high sensitivity, resolution, and flexibility. By utilizing the hydrophilic interactions between oligonucleotides and the stationary phase, HILIC allows for the separation and characterization of these crucial biomolecules. Professionals in genomics, molecular biology, and pharmaceuticals can enhance their oligonucleotide analysis workflows by incorporating HILIC, leading to improved insights and discoveries. Additionally, the minimal effect of HILIC solvents and additives on MS ionization makes it an ideal technique for LCMS analysis.

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