Posted by anne on Friday, October 14, 2011 at 6:30pm.

Hi, I have a question on HPLC (high performance liquid chromatography).
The procedure says to use a
4.6 x 150mm x 3um column.
The relative retention time of Impurity B with reference to the main peak is about 0.5 (i.e the ratio of the retention times is 0.5)
If I use a 4.6 x 100mm x 2.7um column, will the relative retention time of Impurity B with reference to the main peak still be about 0.5?

My calculations below, show yes, about the same, but it has been a long time since I did that. I have asked DrBob to take a look at it to, I am uncertain if he has done any HPLC work.

http://www.jiskha.com/display.cgi?id=1318631421

I haven't done any HPLC work.

To answer your question, we need to consider the factors that affect the relative retention time in HPLC. The relative retention time is the ratio of the retention time of a solute (Impurity B) to the retention time of a reference compound (main peak). It is influenced by several factors, including the column dimensions.

When it comes to column dimensions, the length, inner diameter, and particle size are important. In your case, you have two columns:

1. The first column has dimensions of 4.6 x 150mm x 3um.
2. The second column has dimensions of 4.6 x 100mm x 2.7um.

The length of the column affects the separation efficiency. Longer columns generally provide better resolution, as the solutes have more time to interact with the stationary phase. Therefore, when you switch from a 150mm column to a 100mm column, you would expect some differences in the retention times.

However, it's important to note that the inner diameter and particle size of the columns are the same in both cases (4.6mm and 3um, respectively). These factors play a significant role in determining the retention times. As long as the inner diameter and particle size remain constant, the relative retention time should not change significantly.

So, in conclusion, if you switch from a 4.6 x 150mm x 3um column to a 4.6 x 100mm x 2.7um column while keeping the inner diameter and particle size the same, the relative retention time of Impurity B with reference to the main peak should still be around 0.5. However, some small variations may occur due to the change in column length. It's always a good practice to validate your method whenever any changes are made to the HPLC system.