Overview

Ammonium acetate and ammonium formate are commonly used in HPLC and LC-MS methods because they provide:

• Volatile buffering capacity
• LC-MS compatibility
• Improved peak shape for some analytes
• Enhanced retention control in HILIC methods

These additives are particularly popular when developing methods for polar compounds using HILIC conditions on columns such as Diamond Hydride™, Phenyl Hydride™, Amide™, and Silica-C™.

Solubility Considerations

A frequently overlooked issue is that:  Ammonium acetate and ammonium formate are not soluble in pure acetonitrile.

If a mobile phase contains only acetonitrile, the buffer salts may not dissolve completely and can lead to:

• Particulate formation
• Inconsistent buffer concentrations
• Elevated system pressure
• Column contamination
• Instrument damage

For this reason, an aqueous component is required when using these additives.

Recommended Aqueous Content

A practical starting point is:  90:10 Acetonitrile / Aqueous Buffer

The approximately 10% aqueous content typically provides sufficient water to dissolve common concentrations of ammonium acetate or ammonium formate.

This composition is widely used in HILIC methods and offers a good balance between:

• Buffer solubility
• Polar analyte retention
• LC-MS compatibility
• Method robustness

Using Lower Water Content

Some HILIC methods require stronger retention at the beginning of the chromatographic run.

In these cases, aqueous content may be reduced to:  Approximately 5% water  when appropriate for the application.

However, lower water levels reduce buffer solubility, so care must be taken to ensure that all salts remain fully dissolved.

Buffer Concentration Recommendations

For most HILIC applications, buffer concentrations should generally remain modest.

A practical guideline is:  Do not exceed approximately 10 mM  unless method requirements specifically justify higher concentrations.

Lower concentrations often provide:

• Better LC-MS performance
• Reduced risk of precipitation
• Improved system cleanliness
• More reliable long-term operation

Importance of Mobile Phase Filtration

Whenever mobile phases contain dissolved solids, filtration is strongly recommended.

After preparation:

• Vacuum filter the mobile phase.
• Verify complete dissolution of additives.
• Inspect solutions for visible particulates before use.

Filtration helps protect:

• Pump seals
• Check valves
• In-line filters
• HPLC columns
• LC-MS systems

Even partially dissolved salts can create performance issues over time.

Best Practices

When preparing acetonitrile-rich HILIC mobile phases containing ammonium acetate or ammonium formate:

• Dissolve the buffer in the aqueous component first.
• Add acetonitrile after the salt is completely dissolved.
• Maintain adequate aqueous content.
• Use conservative buffer concentrations.
• Filter all mobile phases before use.
• Monitor for any signs of precipitation during storage or operation.

Key Takeaways

• Ammonium acetate and ammonium formate are not soluble in pure acetonitrile.
• An aqueous component is required for complete dissolution.
• Approximately 10% aqueous content is often a good starting point for HILIC methods.
• Lower water content may be used when greater analyte retention is required.
• Buffer concentrations should generally not exceed 10 mM.
• Mobile phases containing dissolved salts should always be filtered before use.