Phosphorylated compounds can be difficult to analyze by HPLC. However, a wide variety of compounds with the phosphate functional group have been able to be retained with the Cogent Diamond Hydride™ column in ANP mode. Examples include phosphorylated sugars, herbicides, and nucleotides. The strategy can also be applied to other compounds of this type such as isoprenoid phosphates.
If using LC-MS, look for the [M – H]– ions in the EICs (negative mode). For the mobile phase, try Solvent A: DI water + 10 mM ammonium acetate (or formate) and Solvent B: 95/5 Acetonitrile/ DI water + 10 mM ammonium acetate. Then use a standard ANP gradient going from a high %B to a moderate/ low %B. Depending on the retention, adjust as needed to get higher or lower retention. To address any peak shape issues, consider the following three strategies:
• Use a dilute ammonia solution to increase mobile phase pH to 7.0
• Use Teflon bottles instead of glass for the mobile phase to prevent leaching of sodium
• Use a very small amount of EDTA (5-10 microM) in the mobile phase to sequester trace metals, which can cause peak shape problems for anionic compounds such as phosphates
• Use a PEEK column instead of stainless steel