Retention Time Fluctuations (Part 2)

LCMS Troubleshooting Course

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1 - Basics of LCMS Systematic Troubleshooting

2 - Preventive Maintenance Measures

3 - Pressure Issues

4 - No Peaks Detected

5 - Changes in Peak Shape - Part 1

6 - Changes in Peak Shape - Part 2

7 - Ghost Peaks

8 - Peak Area Fluctuations

9 - Baseline Disturbances

10 - Retention Time Fluctuations - Part 1

11 - Retention Time Fluctuations - Part 2

12 - Changes in Chromatographic Resolution

13 - Changes in MS Response

14 - Undesired Fragmentation & Ion Source Settings

15 - Course Summary & Quiz

Part 2: Stationary Phase, Instrument Related Issues and Best Practices

Retention time movement

Retention time is a critical parameter in LC and LCMS, used to identify analytes reliably. They are characteristic of the analyte under certain chromatographic conditions with a specific column, which allows for confidence in analyte identification. Fluctuations can compromise data accuracy, reproducibility, and confidence in analytical results.

This topic is divided into two parts:

  1. 1. Temperature effects, eluent composition, and flow rate issues.
  2. 2. Stationary phase, instrument related issues, and best practices.

This week we are focusing on the issues related to the instrument and stationary phase. 

1. Column & Stationary Phase Factors

Cause Solutions
Stationary Phase Changes
Avoid memory effects by not using columns used with ion pairing reagents for non-ion-pair methods
Recondition or replace columns that are dried out or contaminated. 
Column Equilibration
Allow full equilibration between methods or after storage solvent washout
Column Dimensions
Confirm correct column length, ID and particle size for the method

2. Instrument Related Issues

Cause Solutions
Air Bubble in Pump
Purge thoroughly, especially when switching solvents
This is often associated with changes in pressure. 
Needle Rinse Solvent
Prevent strong needle rinse solvent from reaching the column during injections
Check Valve Issues
Purge with isopropanol to restore check valve function.
Replace the check valve if the issue persists. 
This problem is often observed with pressure trace issues, where the ripple is greater than 2 bar and can look like a saw tooth. 
Sample Diluent Mismatch  
Ensure the diluent matches mobile phase to prevent retention shifts 
Wrong Cycle Time  
If the cycle (or loop) time is not correctly set, the number of data points collected across the peak might be insufficient which can cause the apex of the peak to shift, giving a change in retention time. This is often associated with changes in peak area, and poorly defined peak shape as the majority of the peak is not correctly identified. 

3. Best Practices to Maintain Retention Time Stability

tick Never leave buffers in the system. Rinse them out with pure water (depending on the column) or maintain a low flow rate to prevent salt precipitation.

tick Perform regular system tests to monitor the health of your system and column, ensuring you have a reliable baseline for troubleshooting if issues arise.

tick Control system temperature. Whether for the column, detector, or autosampler, stable temperature conditions are critical for reproducibility and performance. Temperature fluctuations can significantly impact various components.

tick Rinse your system and column regularly to remove contaminants and extend their lifespan.

tick Handle your column with care. It is the heart of your system, along with the eluents. Avoid physical shocks, sudden pressure changes, and operation outside the manufacturer’s specifications.

tick Standardise eluent preparation procedures to ensure consistency and reliability in your analyses.

tickPerform regular system suitability test to check for retention time and resolution

tickDocument retention times and resolution

tickInspect the system for column leaks and worn consumables

 

We have now completed this two part series for the causes and solutions for retention time fluctuation. Next week, we will be looking at changes in chromatographic resolution.

Your Shimadzu LCMS Team

 

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