Chromatography Forum

We have RP-Amide columns which are only lasting 80 or so injections before peaks become too wide (and split) to be useful.
We are running 10mM AF buffer pH3.9 with MeCN .
Injections are 10ul of plasma extracts (in MeCN).

A guard column has made little difference to the column life.

I dont think this is an unusual system? but if anyone has any advice on how to make our columns last longer I'd really appreciate it.


Thanks all

James

James,

A guard column has made little difference to the column life.

Do you change your guard column before the column dies? If so, how often? Perhaps you need to change the guard column every "X" number of injections to prolong the life of the column.

I would think the cause is the irreversible RP column adsorption of some plasma components such as protein and/or lipids, so a better sample preparation might help to prolong the column's life.

if your analyte isn't bound to plasma component, the sample prep usually includes an ultrafiltration followed by acidification/centrifugation, and then LLE or SPE to extract the supernatant. You can use other solvent, e.g. chloroform for the extraction, and then dry it and recondition the residue in ACN for injection.

Also I suggest you to take a look at the SIELC column (www.sielc.com). They claim that plasma can be directly injected into the column without any prep. I've never tried it, but the claim sounds reasonable.

Good luck

I agree with the previous post that the issue could very well be the quality of the sample preparation. An improvement in the sample prep might help. You want to do the MeCN precipitation with a roughly 4:1 ratio of MeCN to plasma. Otherwise, very little gets precipitated and ends up on the column. A much, much better approach is SPE, preferentially with a mixed-mode ion-exchanger, if your analyte can be ionized.

Are you using the same packing in your guard column as in your analytical column? If not, the guard column might not help.

Another possibility is that the amide column is degrading. Some of these columns are prepared in a way that leaves residual amine on the surface. This is on one hand another site for adsorption, on the other hand it could result in column degradation with the dilute buffer that you are using. If this is the case, you can either switch to a modern amide column that does not have that problem, or to a carbamate column (SymmetryShield RP 8/18, XTerra RP18, XBridge RP18) that never had this problem.

I usually got 300-500 injections out of a my column or more. Used 250 ul ACN to 100 ul plasma for protein precipitation. Usually changed my 0.25 u prefilter every 200 injections.

Used a precolumn for a while, but quality poor and often led to degradation of separation. Never had a bad problem with components adsorbed on column from plasma. Did wash the columns and reused sometimes

See more details at

http://users.chartertn.net/slittle/ in matrix effects section.

Good luck.