Poor Equilibration

by | Feb 9, 2021

This Edition of HPLC Solutions is a problem about the different pattern of chromatogram observed within the a few runs due to the lack of column equilibration.

A reader emailed in the following question: I am trying to analyze a peptide (2675 Da) on a 150 x 2.1 mm, 3 µm particle column by LC-MS using an ion trap MS in the positive electrospray ionization (ESI) mode. The first injection is OK, with the peptide eluting at ≈ 15 min with good peak shape, but in subsequent runs the retention time is ≈ 5 min. If I try to run the method the next day, the same pattern occurs. The A-solvent is 10% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA); B is 100% ACN with 0.1% TFA added. The gradient runs from 0-100% B over 30 min, then steps back to 100% A for 20 min prior to the next injection. Can you spot any obvious problems here?

   In cases, such as this, even though a 20 min equilibration time is allowed, I want to check to see if this is reasonable. And while I’m at it, I want to double-check the gradient conditions to see if they look OK, which we’ll look at in the next issue.

HPLC solutions 93-1.jpg

Column Equilibration
Whether we’re running isocratic or gradient runs, I like to allow 10 column volumes of mobile phase for equilibration before running a gradient or when changing mobile phase for isocratic conditions. The column volume, VM, can be estimated as

V M ≈ (0.5 x 10-3) L dc 2   [1]

where L is the column length and dc is the column diameter, both in mm. So for our conditions, V M ≈ (0.5 x 10-3) (150)(2.1)2 = 0.33 mL. So 10 column volumes is ≈ 3.3 mL. The current method uses a 20-min equilibration at 0.1 mL/min or ≈ 2 mL. You should also allow for washout of the system dwell volume (mixing volume) during this process. A well-plumbed LC-MS system should have a dwell volume ≈ 0.3-0.5 mL, so this would add up to 5 more min of equilibration for the present conditions.

   It is pretty obvious that the source of the problem is poor column equilibration. During method setup, it is likely that the column will be run for a sufficiently long time to equilibrate, but by not allowing enough time for the column to equilibrate for the second and subsequent runs, the injection will be made when the mobile phase is too strong, thus reducing the retention time. (You can see an illustration of the actual return to initial gradient conditions in Figure 1 of HPLC Solutions #87.)

   My suggestion is to check this hypothesis by extending the equilibration time to 40 min and see if the results are more reproducible. Since the equilibration of the column is volume-related, it might be more expedient to increase the flow rate during equilibration and shorten the process. For example, instead of 40 min at 0.1 mL/min, try 8-10 min at 0.5 mL/min; then return the flow to 0.1 mL/min prior to the next injection.

This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com

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