What are common causes for less than expected retention times on a reversed-phase LC column? - WKB79531
Article number: 79531
ENVIRONMENT
- Retention shift
- Retention loss
- C18 column
- Reverse Phase
- mobile phase
- care and use
ANSWER
Various factors are possible. See the "Additional Information" section.
ADDITIONAL INFORMATION
Retention loss can be caused by many different factors. Here are some of the more common reasons for lower than expected retention:
- Inadequate column conditioning with ion pairing agent:
- If a method uses an ion pairing agent such as hexane sulfonic acid, it can often take up to 200 column volumes of conditioning in order to saturate the column with the proper amount of ion pairing agent.
- If a column is not conditioned long enough, retention will be reduced until the column is fully conditioned.
- See the PIC Reagents Care & Use Manual.
- Insufficient column re-equilibration (for gradient methods):
- It is recommended to re-equilibrate a C18 column 5-10 column volumes at the end of each gradient table.
- Improper re-equilibration can lead to excess organic left on the column prior to the next injection, which can reduce retention time of the earliest eluting peaks.
- Newer columns may require a longer re-equilibration volume (e.g., 10 column volumes) and older columns may require less re-equilibration volume (e.g., only 5 column volumes).
- Older columns have likely lost C18 ligand (see "Hydrolysis of C18 Ligand" below).
- The particle surface then becomes a little more polar, which allows excess organic to exit the column more easily.
- This allows the more highly aqueous mobile phase to enter the pores more easily, because the pores are less hydrophobic.
- Newer columns have more C18 ligand.
- The particle surface is a little more hydrophobic.
- This makes it more difficult to get rid of residual organic and introduce highly aqueous mobile phase.
- This is why longer re-equilibration may be needed on a newer column, even for an existing method.
- Increasing the re-equilibration to 10 column volumes may help obtain the expected retention time.
- Older columns have likely lost C18 ligand (see "Hydrolysis of C18 Ligand" below).
- Newer columns may require a longer re-equilibration volume (e.g., 10 column volumes) and older columns may require less re-equilibration volume (e.g., only 5 column volumes).
- Pore dewetting (hydrophobic collapse) on C18 columns not designed to use 100% aqueous:
- For methods that require the use of more than 95% water, retention loss can be seen upon turning off the flow and then restarting the flow at a later time.
- Retention time can slowly decrease with each injection as well.
- In both cases above, water is expelled from the pores because the inside of the pores are very hydrophobic.
- Use of at least 5% organic is recommended for traditional C18 columns that are not designed for use in high aqueous.
- See 100% Aqueous Pore Dewetting Test Using Atlantis T3 Columns.
- Hydrolysis of C18 ligand (loss of ligand):
- As a C18 column ages, the C18 ligand can hydrolyze and detach from the particle.
- This results in less retention.
- As a C18 column ages, the C18 ligand can hydrolyze and detach from the particle.
- Column heater too hot:
- If a column heater is not working properly, it is possible that it is hotter than expected.
- Higher temperatures can cause analytes to elute earlier than expected.
- Use an external thermometer to measure the actual temperature of the column heater.
- Flow rate too fast:
- If the system check valves or pumps are not working properly, the flow rate could be faster than expected.
- Faster flow rate will cause analytes to elute earlier than expected.
- Collect waste into a graduated cylinder during a run, for a timed period. Verify that the volume collected is the expected volume.
- Check Valve Problem:
- If check valves become dirty, they may not function properly causing mobile phase to flow towards the solvent bottle, as opposed to towards the column.
- This can a loss of flow and/or an incorrect aqueous/organic ratio.
- If check valves become dirty, they may not function properly causing mobile phase to flow towards the solvent bottle, as opposed to towards the column.
- Inadequate mixing of mobile phase:
- If the mobile phases are not mixed well before entering a column, it is possible to have the incorrect ratio of auqueous:organic solvents.
- Gradient proportioning valve not working properly:
- The gradient proportioning valve (GPV) might be causing too much organic to be delivered, or too little aqueous.
- Either scenario can cause retention loss.
- Mobile phase pH incorrect:
- Ionizable compound retention can change if the pH of the mobile phase in incorrect.
- Ionizable compound retention can also change if the pH of the mobile phase gradually changes over time.
- For a weak acid, pH that is 2 pH units above its pKa (or higher) will make the analyte charged, and more polar in general (less retentive).
- For a weak base, pH that is 2 pH units below its pKb (or lower) will make the analyte charged, and more polar in general (less retentive).
- Incorrect premixed mobile phase composition:
- Having too much organic (or too little aqueous) in a premixed mobile phase will result in less retention than expected.
- Lower Dwell Volume (Gradient Delay):
- A system with a lower dwell volume (gradient delay) will show less retention compared to a system with a larger dwell volume (gradient delay):
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