Factors | Attribute or parameter | Range studied | Optimized data | Proposed range | Purpose of control |
---|---|---|---|---|---|
Mobile phase ratio (Buffer:ACN) | Resolution | 40:60, 50:50, 60:40, 70:30, 80:20 v/v | 60:40, v/v | 60:40 ± 2, v/v | Better separation between peaks, to get > 2 resolution |
pH of buffer | Resolution | 2.5, 3.5, 4.5, 5.5, 6.5 | 4.5 | 4.5 ± 0.2 | Better separation between peaks, to get > 2 resolution |
Flow rate of mobile phase | Resolution | 0.75, 0.5, 1, 1.25 mL/min, | 0.9 mL/min | 0.9 ± 0.2 mL/min | Better separation between peaks, to get > 2 resolution |
Temperature of column oven | Resolution | 0 °C (no control), 10, 20, 30, 40 °C, | 20 °C | 20 ± 2 °C | Effective parameter for changing band spacing and improving resolution |
Organic phase type | Resolution | ACN, MeOH | ACN | ACN | To improve peak shape and resolution |
Degase time | Resolution | 10–25 min | 12–15 min | (12–15 min) ± 2 | To remove air bubble from mobile phase |
Sample volume | Resolution | 5–20 μL | 10 μL | 10 μL ± 0.1 | To reduce chances of more sample loading |
Column type | Resolution | C18, C8 | C18 | C18 | For better separation and resolution |
Buffer type | Resolution | Ammonium formate, ammonium acetate | Ammonium acetate | Ammonium acetate | Ammonium salts are used for LC–MS compatible method |
Buffer strength | Resolution | 5–10 mM | 10 mM | 10 mM | Buffer used to maintain consistent retention, reproducibility, and selectivity |
Detection wavelength | Resolution | 230–250 nm | 239 nm | 239 nm | More absorbance at wavelength maxima |