Date: 14-JANUARY-2016 Last Updated: 30-JUNE-2025
Analyzing phosphorylated compounds by HPLC can be challenging due to their high polarity and tendency to interact with metal surfaces. However, the Cogent Diamond Hydride™ column, used in Aqueous Normal Phase (ANP) mode, offers a powerful solution for retaining and separating a wide range of phosphate-containing analytes.
✅ Compatible Compound Types
The column has demonstrated excellent retention and separation for:
- Phosphorylated sugars
- Herbicides
- Nucleotides
- Isoprenoid phosphates and other phosphate-functionalized molecules
🔬 LC-MS Detection Tips
When using LC-MS, monitor for [M – H]⁻ ions in negative ion mode extracted ion chromatograms (EICs) for optimal detection of phosphorylated species.
🧪 Recommended Mobile Phase Conditions
Use the following mobile phase system for ANP:
- Solvent A: DI water + 10 mM ammonium acetate (or ammonium formate)
- Solvent B: 95:5 Acetonitrile:DI water + 10 mM ammonium acetate
Start with a high %B and apply a gradient to a moderate or low %B to adjust retention. Fine-tune the gradient based on your compound’s behavior.
🛠️ Troubleshooting Peak Shape Issues
To improve peak shape and reproducibility, consider these strategies:
- Adjust pH: Add a small amount of dilute ammonia to raise the mobile phase pH to ~7.0.
- Avoid Sodium Contamination: Use Teflon bottles instead of glass to prevent sodium leaching.
- Metal Ion Interference: Add 5–10 µM EDTA to the mobile phase to chelate trace metals that can distort peaks of anionic compounds.
- Column Hardware: Use metal-free, coated HPLC columns to avoid unwanted interactions with stainless steel surfaces.
📦 Ordering Information
Clicke HERE for ordering the Cogent Diamond Hydride™ column or to learn more about its specifications and available formats, please visit our [product page] or contact our technical support team.
