Serum sample preparation for LC injection

[skilly]

Hi!

I'm using HPLC since one month.
I have to inject human serum samples, but I have high back pressure (4.6-5.9 kpsi) even with 20 ug of protein depleted from albumin with IgY system (Beckman coulter).
I have to wash the column with acetonitrile 100% for 30 min so that the pressure return nearly 2-2.2 kpsi (the top for a new column).

my column is a C18 non-porous reverse-phase.

The serum is at -80°C. Spin 10 min at 13400 rpm, deplete 20 ul (as protocol) of serum with Igy (recover 1 ml), protein assay, solubilization with urea buffer (TSU), centrifugation 30 min at 14000 rpm (20000 g).
But the column have high back pressure when I inject this sample....
How I can make?

Thank you!!!

[HW Mueller]

Looks like I am not the only one who doesn´t know what the IgY system does, etc., etc.
Anyway, I will try: Assuming that mobile phase (what is it?) does not remove the backpressure (really have to run ACN?), one can say that it´s highly unlikely that the backpressure is due to a protein (ACN has a tendency to precipitate most proteins) nor that viscosity of the sample is the culprit (would be a temporary affair), or that particulates, unless they dissolve in ACN???, are behind this. Thus to get an idea about the cause one would have to find out exactly what is in the material that is injected.

[skilly]

Thank's for your suggest and sorry for the omissis information.

I use a gradient, with 0.08% TFA in ACN (B) and 0.1% TFA in water (A).
Probably the column is overloaded due to a non denaturated protein (my personal error!!! the first time that I have injected serum, I didn't solubilize in urea the proteins...) and it can't be clean up again..

Now my question is: for serum sample is better a C18 column non porous or porous? or another type of column?

Best regards,
Manuela

[HW Mueller]

Now you gave me the rest....am utterly confused. The ACN doesn´t restore the column after all? (It is highly likely not to help if a protein was involved). Etc.
My suggestion: Read some books on protein purification, analysis first.

[skilly]

I had overload the column the first time with unsolubilizated proteins; then I clean up with ACN also in reverse flow and the pressure came back to optimum.
However, when I inject the serum solubilizated correctly (as in literature) is just the same, high back pressure.

I clean up also with isopropanolo 100% and urea 6M (I repeat a lot of injections to understand the problem.... the column or the sample?).
My hypothesys is that I can't remove the proteins non denaturated from the column.

Now I'm thinking if this type of column is appropriate for my sample.... I find this is, but I need an opinion, before to use a new column with the same samples.

thank you

[HW Mueller]

If you really ran ACN into a column loaded with proteins you may really have a problem. I don´t think you could get rid of it via urea. You may need something like detergents (3w% Li dodecylsulfate + 0.025M dithiothreitol, the latter to reduce possible -S-S-). Even if that restores your column you still have to know whether your injections include substances which plug a column.