Chromatography Forum

On the very first 3-micron column that I used in my life, the backpressure increased 3-fold within 24 hours of injecting standards (this was at some point in the early 1980s). Based on this, I concluded that the technology was not quite there yet. Over time, we have learned how to do this better.

Also, the inversion of a column with a wide frit at the column inlet is not an immediate disaster. If you hook the column to the detector for flushing it (which is not the recommended procedure), you get a sharp air-bubble-type signal every half minute or so. With other words, the packing is not pouring out of the column in a hurry.

There is still the question about whether anybody has injected some difficult matrixes (including sticky proteins) on these "unsymmetric" columns. No plugging in both directions (since a large portion of the bed was crudded up)?

Somehow intuition would tend to indicate that it might be more prudent to do this unsymmetric thing with different frits only on a guard column.

to jduan: if changing the flow direction can make the bed shift, then the column was poorly packed, period.

to the column manufacturers: if the recommendation is to use an in-line filter anyway, why not make both the inlet and the outlet frits on the column 0.5 micron?

Because doing so would cut into their guard column sales?

I am taken aback that the inlet & outlet frits are no longer the same. That said, I must confess that I have generally found that reversing a column at either end of its useful life has made little difference in its resolving performance or its back pressure. I just replace them, but then I have 1) fairly clean samples, 2) columns that generally cost <$500, 3) some columns with several thousand injections on them that are still OK. I tend to shy away from brands that exhibit back pressure problems before other types of problems, other things being equal.

Hans, we routinely inject difficult matrices on these non-symmetrical columns. I am not aware of any problems that could be attributed to the lack of a fine filter on top of the column. I actually just looked at a lifetime study on a 3.5 micron column using a MeCN-precipitated plasma sample without a guard column. The column looked fairly unchanged after 1000 injections, with an only marginal increase in backpressure.

Uwe, did you have an in-line filter upstream of the column (I surmise "no" from context, but . . .)?

.. and does the 3.5-micron column also use "asymmetric" frits (different porosity on inlet and outlet)?

Ah yes, Uwe, an ACN precipitated sample! Most of the horrendous problems we had was with restricted access columns, where you are supposed to inject plasma/serum. Later I never injected anything, anywhere, unless at least most of the proteins were precipitated with Na2SO4, org solvents, or "removed" with ultrafilters, or a combination of these. Still with some catabolic patients we had problems even at the third column (in series, commonly and strangely called three dimensional). I mentioned this before: such plasma/serum even plugged 0.45µ microfilters!
When we learned to put the right amount of solid Na2SO4 to each plasma/serum sample the problems almost vanished.

Tom

In this experiment, no inline filter was used and yes, this was an example of an asymmetric column with the larger filter at the inlet.