Help on: Retention Time Variability

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Question:
“I have a question regarding retention time variability between different types of HPLC systems. An experiment was performed on an Agilent 1200 HPLC and on a Waters 717 instrument using a gradient profile. On the Agilent, the retention time was 14.7 minutes, and on the Waters, the retention time was 19.6 minutes. The same mobile phase was used for each system along with the same sample preparations, which seems to point to a problem with the Waters instrument (the Agilent gives typical results). What are some issues that could cause such variability with the retention times?”

Answer:
“The most probable reason for the difference in retention time that you are observing is that the dwell volume for the two HPLC systems being used is different. The dwell volume relates to the time taken for a change made in the mobile phase proportions to reach the column. The dwell volume for modern instruments like the Agilent 1200 is quite small (typically less than 1 mL) but for older instruments it can be in the region of 2 to 3 mL or larger. The Waters 717 is an autosampler but I assume that it is connected to other Waters components of a similar age. It is likely that the dwell volume for this system is greater than that for the Agilent and thus is the cause of the larger retention time.

You can easily check if the dwell volumes are different by determining this value for each system. I have provided directions for this in a previous blog post. You may also be interested in the reply to another MTS Helpdesk question which was similar to yours where I discuss further the reasons for differing retention times for different systems.“