Bonjour, j'ai une question si quelqu'un a une réponse. J'ai une colonne Uptisphere Strategy 100 Å Bonding HILIC-HIT avec une taille de particules de 3 µm et une longueur de 100 mm. Je n'arrive pas à séparer le glycérol et le sérinol sur une HPLC avec deux détecteurs, UV et RID, en mode isocratique. Ma phase mobile est un mélange d'acétonitrile/eau (85/15). Même en changeant ce ratio et aussi le pH, je n'arrive toujours pas à les séparer, ils apparaissent au même endroit.
Separating glycerol and serinol on an HILIC (Hydrophilic Interaction Liquid Chromatography) column can be challenging, especially if they have similar hydrophilic properties and retention times under your current conditions. Here are some strategies and considerations to help improve the separation:
Adjusting Mobile Phase Composition
1. Mobile Phase Composition: In HILIC mode, the ratio of acetonitrile to water is crucial for separation. Since both glycerol and serinol are polar compounds, slight adjustments can significantly affect their retention times.
· Increase Water Content: Try increasing the water content of your mobile phase. For example, adjust to 90% acetonitrile and 10% water, or even 95% acetonitrile and 5% water. This change can alter the retention times by modifying the interaction between the analytes and the stationary phase.
· Buffer Addition: Adding a small amount of a volatile buffer (e.g., ammonium acetate or ammonium formate) to the water component (typically at low concentrations, around 5 mM) can enhance separation by promoting ion pairing interactions or modifying the pH slightly.
Gradient Elution
1. Gradient Elution: Implementing a gradient elution instead of isocratic mode can sometimes improve separation. Start with a lower water content in the mobile phase and gradually increase it over time. This approach can help in separating closely eluting peaks by varying the polarity during the run.
Temperature Optimization
1. Temperature Control: HILIC separations can be sensitive to temperature. Try adjusting the column temperature slightly (within the recommended range for your column) to see if it affects the separation of glycerol and serinol.
Detector Sensitivity and Detection Wavelength
1. Detector Sensitivity: Ensure that both UV and RID (Refractive Index Detector) are optimized for detecting glycerol and serinol. Adjust detector sensitivity and optimize the detection wavelengths to enhance the detection of your compounds.
Column Selection
1. Different Column Type: If possible, consider trying a different HILIC column with a different stationary phase or different particle size. Sometimes, changing the column chemistry slightly can provide better separation for specific analytes.
Sample Preparation
1. Derivatization: Consider derivatization of glycerol and serinol to improve their chromatographic properties. Derivatization can change the polarity and improve retention time differences, making separation easier.
Method Development
1. Systematic Approach: Systematically vary one parameter at a time while keeping others constant to identify the optimal conditions for separation. This approach helps in understanding the interaction between the analytes, mobile phase, and stationary phase.
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