What is Peak Retention Variation?
Before we can troubleshoot an issue, it's essential to understand the basics. In the inaugural installment of our series, we will embark on a journey into the heart of chromatography to demystify Peak Retention Variation. What factors contribute to the shifting retention times of analytes during an HPLC run? How do temperature, mobile phase composition, and column characteristics influence this phenomenon? We'll explore the theoretical underpinnings, examining the role each variable plays in shaping the chromatographic landscape.
Troubleshooting HPLC Peak Retention VariationFocusing on troubleshooting strategies to tackle Peak Retention Variation head-on. From systematic diagnosis of the issue to targeted adjustments of critical parameters, we'll guide you through a step-by-step process to restore chromatographic precision.
We unravel the enigma of Peak Retention Variation and empower analysts with the knowledge to optimize their HPLC methods. As we navigate through the theoretical foundations and hands-on troubleshooting techniques, our aim is to equip you with the tools needed to conquer the challenges of chromatographic separations and elevate your analytical prowess.
Troubleshooting HPLC Peak Retention Variation
Possible Cause | Prevention/Solution |
Decreasing Retention Times |
Loss of bonded stationary phase | Replace column Operate at pH 2-8 for silica based RP columns |
Active groups on stationary phase | Use organic modifier in mobile phase Increase buffer strength |
Increasing flow rate | Check and adjust pump flow rate |
Column overloaded | Reduce amount of sample injected Use column with larger internal diameter |
Increasing Retention Times |
Changing mobile phase composition | Cover solvent reservoirs Prepare fresh mobile phase |
Loss of bonded stationary phase | Replace column |
Decreasing flow rate | Check and adjust pump flow rate Check for leaks in system, including pump seals |
Bubbles in mobile phase | Check flow rate and pressure Degas mobile phase |
Fluctuating Retention Times |
Insufficient column equilibration | Equilibrate column longer between runs Condition the column with concentrated sample |
Change in mobile phase composition | Check the make-up of mobile phase and make up new if necessary Check proportioning-valve accuracy |
Insufficient buffer capacity | Use buffer concentrations >20mM |
Fluctuating column temperature | Stabilize ambient temperature Thermostat the column |
