Chromatography Forum

Hello,

We have made some protein precipitation tests from human plasma.
In some samples seems that proteins were not fully precipitated by the organic solvent used (methanol w 0.1 % HCl), because the amount of the solvent was too small.
The pressure on the column begun to rise (cca. 80 bar after 20 samples) and remains at cca. 250 bar (MF MeOH/Water 1/1).
Now my question is: Is it possible to remove (solve) the denaturated/precipitated proteins stacked on the column by means of a proteolytic mobile phase, or the column is dead?

Best Regards

Dear Totosr,

I am very positive you can regain your column. There should be a way. However you did not specify what column you used. I suppose you are using a C8 or C18 column?

Regards,
Daniel.

Dear Daniel,

I have used for the mentioned tests a Zorbax Rapid Resolution SB-C18 2.1*30/3.5u column.
The column is still in use, but as I specified shows an increased backpressure of 250 bar. If you have an idea how could I regain my column...
Peak shape, reproducibility of areas still ok.

Best Regards,

TotosR

This also has been discussed many times. For extreme cases we have used a mixture of lithium dodecylsulfate and dithiothreitol dissolved in water. Sometimes it is sufficient to use chaotropic substances like some salts, or 6M urea, or even just lots of mobile phase, maybe with a different amount of organic modifier.

Dear TotosR,

in this case I can only agree, I would use the following washing sequence:
(all flushing should be done with at least 10 column volumes)
remove buffer salts from column -> use ACN or MeOh and water 50/50.

next wash at high organic modifier conentration,
next wash with different organic modifier (e.g. go from water to THF, to Methylene chloride, back too THF then to normal mobile phase. ALWAYS make sure that the modifiers are miscible with each other. If not, use 10 column volumes of an intermediate modifier which is miscible with both.

If you have problems with pressure (especially when using MeOH) use a lower flow initially, increase back to normal flow over time if possible.

If this does not help, use 6M Urea, but again be careful not to precipitate the Urea on the column when changing from a 100% organic phase that precipitated Urea.

This procedure will work with many of our columns, but obviously I can not give any guarantes for columns of other manufacturers.

Best regards,
Daniel.

Thanks for your answers I'll try to pump mobile phase first than to take urea or dodecylsulphate when still no result.

Best Regards,

TotosR

If I had protein precipitation problems, I wouldn’t mess with pure organic solvent or pure water – neither with any mixture of these two – without some low concentration buffer or an acid.
Alternatively; urea or dodecylsolphate should be the easiest and the safest way to go, unless you know more about your protein/s (e.g. pI’s etc.)

Best Regards

Hello,

I have tried the cleaning procedure with 6M urea solution without results.
Moreover after washing with 75 ml urea solution followed by washing with HPLC water ca. 100 ml the pressure on the column at 0,3 ml/min was over 250 bar!
Another problem has appeared after the HPLC system was used for the cleaning procedure.
I wanted to run some samples with MS (ESI) as detector. I have changed the column to a good one. After ca. 30 min equilibration a have injected a sample and the signal was much below the yesterdays level for the same control sample. Then I have opened the source and seen that a very fine white powder in deposited in the whole ionization chamber
I have cleaned the chamber with ultrapure water but after a couple of minutes the white powder was back. It seems to block also the capillary.
I have changed the mobile phase flask back to its original after cleaning the column, and the used MF was collected in a flask during the procedure to avoid MS contamination with urea.
When using solvent from the other channel of the binary pump (but MeOH as mobile phase - tomorrow I'll try to put water on the other channel) no white powder was formed.
My question is, that is possible that urea was stacked somewhere in the HPLC system, and dilutes slowly and passes to MS. My idea is that the degasser could be the wicked peace in the whole system.
Please HELP, to get back my MS without wenting, and to get rid of the contamination.

Beste Regards,

TotosR