Chromatography Forum

danko,
did you agree with the daily column washing or it depend on the case and what about my case , and what about the case If I use Agilent column.

HW Mueller,
did you suggest raising the buff conc. to 0.1M for example or to change its type? and dose this help in solving problem

I start with retintion time Of 7.40 and it decreasing within week to give 6.0 , it decrease only
I injecte the supernatent directly (there is aguard column) and inject about 50 samples daily

Best Regards

Your guard column isn´t going to alleviate any effects due to perchloric acid, incidentally, neither is it going to prevent rt changes due to matrix adhering to stat phase.
Rather than changing the buffer concentration I would get rid of the perchloric in the samples to be injected.
One reason for asking how many times you have seen this shift is that I hoped to also find out whether anything had restored the original rt at some point.

Hans, I disagree...

The guard column will dilute the acid before it hits the main column, and it will capture strongly adsorbed constituents of the matrix before it gets to the column. That is the purpose of a guard column.

"it will capture strongly adsorbed constituents of the matrix before it gets to the column." - I agree with Hans that retention can decrease and it is exactly because of what you said. Guard column adds to retention and when it captures "strongly adsorbed constituents of the matrix" retention will decrease

it's funny that discussion continues and we still got no information whatsoever about steps taken to prevent RT decreasing and their results

That goes for perchloric also: If it kills enough column without a guard it will kill enough stat. phase in guard + column to have the ~ same effect. As a matter of fact, a guard column doesn´t prevent plugging either, it just simplifies the correction.

grzesiek, a discussion about whether a guard column will stop the rt shift or not is right on the ball.

You guys missed the point: if these events happen, they affect the guard first. You see a change, you replace the guard, and you are back in business. If not, something else is happening...

danko,
did you agree with the daily column washing or it depend on the case and what about my case , and what about the case If I use Agilent column.

Hi Smkh,

Regarding the daily column wash I’m completely in line with Uwe (i.e. must be case sensitive )
A single advice – if I may: Proceed with the column test (i.e. simply dismount the Phenomenex and mount the Agilent) and leave the rest to subsequent reflections.
The more parameters one alters simultaneously, the more expertise is required to evaluate the results!

Best Regards

Uwe, I assumed that one would choose a guard which has negligible effect on plate hight etc., so the changes as discussed will be negligible as well for practical purposes.

"if these events happen, they affect the guard first. You see a change, you replace the guard, and you are back in business" - this was my point exactly, but we don't see Smkh post any results of changing anything (as for now)

At least we seem to agree that a guard does not change the effect giving rise to the rt change, unless there is a miraculous stat phase in the guard.
Now if one assumes that the effect is progressively moving down the column (and guard) it is imaginable and reasonable that the effect is still limited to this guard (if it is at least 2mm long, column being ~20cm) after a change of about 0.1 min of rt shift. To stay within 0.1 min, very roughly about 10 guards would be needed for one week for the described case.

HW Mueller, Uwe Neue
About guard column, I change it continuously without restored the original tr, I never get the original tr whatever I do, column washing or guard changing.

grzesiek,
I will follow and discuss the results of any steps you will suggest to prevent this problem.

Danko,
I will try Agilent soon and you make me think in away to keep it available in my stock.

"I will follow and discuss the results of any steps you will suggest to prevent this problem. " - ok I thought it's gonna be "only theory" discussion

"About guard column, I change it continuously without restored the original tr, I never get the original tr whatever I do, column washing or guard changing. " - I know there were some issues withstability of first gemini, have you tried to replace the column?

My guess: You ruined your column by continuously injecting a highly concentrated perchloric acid. (What is the molarity you use, how much volume on how much sample to precipitate?)

I use 20% perchloric acid
then take 50 microliters to 0.500 mL plasma