Buffers are used in HPLC for several reasons:
1. pH Control:
Buffers help maintain a stable pH level in the mobile phase and lock the pH, which is essential for:
-Ionization of analytes: pH affects the degree of ionization, influencing polarity and interaction with the stationary phase.
- Stationary phase stability: Some columns are sensitive to pH changes.
2. Peak Shape and Resolution:
Consistent pH of buffers can improve peak shape and resolution by:
- Reducing peak tailing
- Increasing column efficiency
- Enhancing separation of analytes
3. Reproducibility:
Buffers ensure consistent pH conditions, which is crucial for:
- Method development and validation
- Repeatable retention times and peak areas
- Reliable quantitation
4. Column Protection:
Buffers can help protect the column from:
- pH-related degradation
- Ionization-induced damage
Common buffers used in HPLC include:
- Phosphate buffers
- Acetate buffers
- Formate buffers
- Ammonium buffers