Retention Time (RT) Shifting or RT Variation in HPLC analysis

This is one of common problem seen in HPLC analysis. 

To find out root cause of any problem, you need to identify exact behavior. 

Need to identify whether RT is continuously varying or eluting earlier or later. 

Also need to check whether RT variation occurs in Isocratic Method or Gradient Method or in both. 

Moreover, you can check whether RT shifting happens for one specific compound peak in chromatogram or for all peaks. 

Let’s take an Example for one of Specific Product Analysis: 

For example: 

Case 1: For one of Product analysis, expected RT for Compound peak is 5.5 minute. For multiple Injections, if this expected Peak is eluting at 4.6 minute, in some injection 4.8, in some injections 5.8, 5.9, 6.0 like this. Looking at this different RT, we can say it is continuously varying.

Case 2: Now, if Peak RT is varying like this for multiple injections: 

If peak is eluting earlier gradually run by run, like first run 5.5, second, 5.4, third, 5.2 and so on or gradually delaying. This case, RT is eluting early or delayed.

Above two cases describe problem behavior, let’s see probable causes which is generating this problem.

• Peak Elution depends on Flow, Mobile Phase Chemistry, Column Chemistry, System equilibration done properly before starting analysis and Temperature etc. Check basic functionality of HPLC.
• Make sure Instrument Method setup is proper, Mobile Phase preparation, different solvent mixing, properly degassed Mobile Phase, Lab temperature is proper before jump to HPLC system Hardware. 
• If you are using Solvent Compressibility and/or Stroke Volume Parameter in HPLC Pump Parameter, make sure it is set correctly. 
• Sometime if Temperature is not proper or Column Temperature is not stable or varying, it might cause RT variation. 
• Check whether you have replaced or changed any Capillary in HPLC system, and you are facing RT issue after this activity. If you will not use Standard tubing’s/capillaries it will impact Delay volume. Change in Delay volume can cause RT variation in Gradient Methods. 
• If Pump Flow is not proper, it can cause RT shifting run by run depends on Mobile Phase and Gradient composition also. 
• If flow is continuously varying it will cause RT variation. Also, you can check Pressure plot for each run which will give you an idea of flow variation. Flow variation will cause pressure fluctuation also. Comparing Problematic Pressure Graph with earlier good analysis pressure graph will give you some clue. 
• Improper flow can be caused by HPLC Pump component functionality like Inlet/outlet valves, pump drives, pump other components. 
• Check that HPLC degasser is working properly. 
• Sometime, Gradient Valve, usually called GPV (Gradient Proportional Valve) or MCGV (Multichannel Gradient Valve). If This Valve is not working properly, it may cause RT issue. Gradient valve mul-functionality can give you stable pressure in some cases but still RT variation happens. 
• If all the things are working well, you can check whether this RT variation occurs specific to one product analysis only or for all products. 

All above steps are given to have an idea to find out probable or root cause. There can be different reason for RT variation issue apart from given above. Appropriate solution need to be applied once root cause is identified. Also it might need engineer visit to resolve instrument issue.