Chromatography Forum

Hi, we have a Waters e2695-2998 system in our analytical laboratory, and the column that we use is:
Waters SunFire, C8 3.5um, 4.6x160mm.

Given the number of injections and some slight peak splitting problems observed in some chromatograms, I changed to a new column with the same brand, same product number. Before I start doing any injection, I flushed, and equilibrated the column according to the use manual.

After that, I started to run some samples from different project. I found that the retention time is 0.2-0.3mins consistently later than the retention time from the previous column.

I never use a guard column on our system, but I do use precolumn filter and it is the same precolumn filter, same fittings from the previous column.

I don't know what is the reason causes the 0.2mins delay. I would much appreciate people's help!

Thank you All!

A certain amount of column-to-column variation is normal (that's why you run standards). Factor in any changes in surface chemistry from use, and you are lucky they are thst close!

Tom is right.
I would like to ask you what do you know about history of the previous column. Used buffers, pH values etc. and how many injections you had on the previous column. Do you use one dedicated column for one specific method or do you use one C8 or C18 column for different methods/projects?
These are only a few parameters to influence RT on a column over the time. As Tom said, you have only a tiny little RT shift. Column temperature is constant?

Given the number of injections and some slight peak splitting problems observed in some chromatograms, I changed to a new column with the same brand, same product number. Before I start doing any injection, I flushed, and equilibrated the column according to the use manual.

After that, I started to run some samples from different project. I found that the retention time is 0.2-0.3mins consistently later than the retention time from the previous column.

I don't know what is the reason causes the 0.2mins delay. I would much appreciate people's help!

Common. Expected.

With use, you either had some packing dissolve and/or some of the "active sites" have adsorbed stuff, so less separating/retaining ability, so the peaks gradually eluted sooner as your first column aged.

If me, I certainly wouldn't worry about it.

If Curly Howard, I "soitenly" wouldn't worry about it.

Agree with the previous posts, a 0.2-0.3 minute change from column to column is certainly reasonable.

If Curly Howard, I "soitenly" wouldn't worry about it.

Thanks for the Friday morning Three Stooges reference. Good way to start the day.

If Curly Howard, I "soitenly" wouldn't worry about it.

Thanks for the Friday morning Three Stooges reference. Good way to start the day.

Jerome Lester Horwitz, better known by his stage name Curly Howard, was an American comedian and vaudevillian actor.

Thank you everyone for the reassurance, apart from 0.1% TFA, I have never used any other buffers in our system so far. There were over 10000 injection had been done in the previous column...I acutally don't know how often do I need to change the column, the reason I changed the previous column is because I started noticing some minor peak splitting in an organometallic compound, but every other compounds are still fine. In addition, we can't afford to use one column for one specific method, in our case we just use C8 column for all different methods.. Our column temp is always at 40 degrees.

Thanks everyone again for your prompt response!