Chromatography Forum

The issue is that you are injecting a badly prepared plasma sample...
Under those circumstances, a lot of things will end up on the column, reducing retention, changing selectivity.
You have two options:
1. Improve the sample peparation to reduce the gunk that you are injecting on column. I can give you advice how to do that (SPE).
2. Live with it and throw the column away when you are not happy with the results any more.

It´s a bit less concentrated than I thought, but still, you have a bit lower than pH = 1 to start (at injection), the buffer will chew up a small bit, there is a bit dilution (not that much, I expect the H+ to move out at to). I would not discount the idea as yet, but would also still keep deposits, or similar, on the column in mind. Thus I wonder whether you can resuscitate the column with chaotropics or even detergents.

Uwe Neue,
SPE is recommended and widely used in many applications but unfortunately my lab dosn't have agood one, what we have is asmall , manual one which is not useful to prepare many batches daily.

HW Mueller,
is the used perchloric acid as described is aharsh?! If I have to use it what is the suggestions to reduce its effect.

what I see that column brand and sample preperation is the main issues in this case and in all methods development generally, which led me to ask all experts from their own experience about two things:
*name of robust and stable column brand.
*advantages and disadvantages of SPE , is SPE really the magic solution for many sample preperation problems.

Let us go back to the beginning: It appears that you are never doing any wash of the column. Is this (still) correct?

With respect to SPE, you do not need complex equipment, just a vacuum line and an extraction manifold with a pressure gauge. For the processing of many samples, you can work with a 96-well plate to process the samples in parallel.

Here are some references on SPE:
U. D. Neue, C. R. Mallet, Z. Lu, Y.-F. Cheng, J. R. Mazzeo, “Techniques for Sample Preparation Using Solid-Phase Extractionâ€

One neutralizes acid by adding some base.
If you do an SPE and don´t use perchloric in your eluent you will be rid of perchloric sort of automatically.
If I saw this correctly then you said that washing with organic solvent, etc., did not rejuvenate the column. That is why I suggested the more drastic measures (directed at macromolecules). If this improves your column you will also have a good proof that you ned a better cleanup.

pH adjustment should be made consistently, whatever the method be.

I see a lot of problem with your method. First adjusting the pH after adding the ACN, and then filtering it.

I would:

adjust the pH of the aqueous part first, followed by filtration to get rid of any buffer particles (undissolved impurities) - and then add the required organic.
This is to ensure any column plugging, and organic evaporation during degassing filtration.

Your buffer capacity is zero. And if your compound pKa is close to 3.0 pH - then you can assume a drastic RT shift with minor pH changes.

Overall, very poorly drafted method.

I will blame the Phenomenex later. Contrary to what people say, I had the best performance with Phenomenex columns. And so do, ACE, Agilent and Waters brands.

Hi Mohan

Firstly, the buffer capacity, in this case, is not zero. If the pH was above 3.2 then there would have been a justification for your statement.
Secondly, how can persistently decreasing retention times that can not be restored to the initial state be the result of pH variations upon injection?

Btw. the degassing procedure is most effective when it’s carried out following the mixing of aqueous and organic liquids and not prior to that.

Best Regards