Chromatography Forum

Hi all,

I am doing reversed phase LC. I have had a sudden retention loss - by a couple of minutes.

We had an issue with the solvent running dry last week - i know, not the best. i rehydrated the column over a few hours at high flow. I re-ran my control sample and it was fine. no change in analysis.

ccleaned the column and took it off to do other analysis on a different column. reinstalled last week and thats where the sudden RT shift came.

Can this really be due to the column or am i looking at something else?

What is unusual is i run a different method on the same column - same mobile phase but different gradient and flow. And this showed no RT shift at all. Any suggestions at what i'm looking at? I'm worried its the column but i think it may be a method/configuration issue with not seeing any difference on the same column but different method.

thanks in advance!

First thing would be to check for leaks.

NO possible way to know as too much information is missing. If you have a brand new (spare) column to swap in and test, that might provide the quickest answer. And.. you should always have a new column available to use for any important methods as time = money.

However, to troubleshoot any retention time drift or variability problem, the common areas to address are the same. Here is a link to a free article that may assist you to troubleshoot this and other related problems. "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it"; https://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html

Do you have an Agilent Quatenary pump? If so it can be the proportioning valve giving inaccurate gradient. If any part of the gradient is 100% and one of the valves is leaking then you won't have 100%.

Another problem I have had is if the RT shifts early, check the inlet filter at the mobile phase bottle, especially on the aqueous phase. If any algae grows in the aqueous bottle it can clog the inlet filter and slow the flow of that mobile phase enough to shift the percentages in the actual outlet to the column.

James_Ball wrote:
Do you have an Agilent Quatenary pump?

We always purged each channel at start of day 5 min. at 5ml/min even if we were not using all four.

hi all

yes i have a quat pump and all 4 lines are purged at the beginning of the day.

I jsut find it unusual that for the same column but a different method is see no RT shift. same eluents too. just a different flow and gradient.

I've checked for leaks but nothing.

thanks!

Kate,

In that case check your 'proportioning valve' that supplies all 4 channels. As I recall the verification test uses 100% Acetone and 100% Water. Be sure your column is offline. As I recall, the response time should be much less than 1 minute.

Once the test is completed satisfactorily, installed the column and check for leaks (especially around the column). The Agilent 1090 and 1100 had ~4 leak detectors.

Check for leaks at the fittings using a tissue. Leaked mobile phase can evaporate before it even gets to the sensor.

HPLC chemist wrote:
Kate,

In that case check your 'proportioning valve' that supplies all 4 channels. As I recall the verification test uses 100% Acetone and 100% Water. Be sure your column is offline. As I recall, the response time should be much less than 1 minute.

Once the test is completed satisfactorily, installed the column and check for leaks (especially around the column). The Agilent 1090 and 1100 had ~4 leak detectors.

I often did a check by placing all 4 lines in 100ml graduated cylinders then pumping at 25% on all four lines with a flow of about 4ml/min. After 20 minutes each cylinder should drop 20ml, any difference is a difference in the proportion of each line versus theoretical.

We always flush the lines, but if one of the metering valves is leaking a little it can cause a shift in the % composition of the gradient. I saw it most when it was near 100% for any component, closer to equal % and it didn't matter as much.

hi everybody thanks for your replies!

it seems i had a leak. i fixed this but i still have a 1 min RT change now. Still something isn't right.

I changed tubing from the column exit to the DAD for a shorter one. How much can i expect RT to change if i halve the tubing length between column and DAD? surely not 1 minute?

kate

kate_m wrote:
it seems i had a leak. i fixed this but i still have a 1 min RT change now. Still something isn't right.

Good. maybe there were two leaks !

kate_m wrote:
I changed tubing from the column exit to the DAD for a shorter one. How much can i expect RT to change if i halve the tubing length between column and DAD? surely not 1 minute?

Good not to have extra lengths of HPLC tubing. But such shortening will not affect the RT hardly at all, assuming that the tubing is HPLC tubing of same or smaller i.d.

Some folks have mistakenly used wider i.d. 1/16-inch tubing !!