Chromatography Forum

It has been suggested that HPLC columns be conditioned by repeated injections of an inexpensive (sacrificial) protein (to block sites on column/hardware that would otherwise irreversibly bind low-abundant sample).

I used beta lactoglobulin to condition a 1000 A 1 x 150 mm PLRP column. I almost certainly injected too much, and now I cannot get rid of it. I have done countless up-down gradients (5-60++% MeCN/0.1% TFA) and back flushes with no luck. I've changed pre-column frit, and connections. A blank run yields a super clean baseline plus this artifact (which shows up as a beautiful looking peak eluting at approx the same MeCN%).

The size of the peak decreases little, if any, with successive blank gradients.

Could this protein have precipitated at the inlet frit, causing it to bleed off over time?

Are you sure that the peak is protein?

I'm almost certain the peak is protein. *only* peptide/ protein samples have been injected on that column. The artifact absorbs at 220 as well as 280 nm.

Does it sound as though the material is located on the column, or at the frit?

I've gathered the following cleaning protocols:

1 M NaOH
1 M HNO3/IPA (50/50)
70% HCOOH (A) and IPA (B), gradient from A to 100% B.
40/40/10 MeOH/chloroform/1% aqueous HCOOH, with repeated injections of concentrated HCOOH

Assuming the contaminant is beta lactoglobulin, which would be best?

Any problem introducing HNO3 to the support with aromatics?

It looks like you are introducing the stuff with each injection. A deposited source should decrease with the number of blank injections, unless you have an endlessly large deposit. Absorbance at 220 and 280nm does not prove that it´s a protein, on the other hand, it is also possible that the blanks are contaminated with a protein.

Where did you find these cleaning protocols? Your column is not silica based?
Did you get a "clean" baseline before you injected the protein?

It looks like you are introducing the stuff with each injection. A deposited source should decrease with the number of blank injections, unless you have an endlessly large deposit. Absorbance at 220 and 280nm does not prove that it´s a protein, on the other hand, it is also possible that the blanks are contaminated with a protein.

Where did you find these cleaning protocols? Your column is not silica based?
Did you get a "clean" baseline before you injected the protein?

I've ruled out mobile phase contamination since freshly prepared 0.1% TFA, MeCN (using HPLC water, TFA) did not change things.

I haven't done a single injection on this column since the original beta lactoglubulin several blank runs ago. I think it -is- an endlessly large deposit.

The PLRP (polystyrene divinylbenzene) column is not silica based, which should permit a more aggressive cleaning protocol than silica-based RP columns. I think this is what I will have to resort to.

What is the UV spectrum of DVB?

What is the UV spectrum of DVB?

From quick google search.. not sure how reliable..

If DVB = divinylbenzene then Uwe is getting at bleeding as possible source. I also googled and found this:

http://135.196.210.195/suppdata/CC/b8/b ... 17598e.pdf

This is totally different than the spec above, it has strong absorbance at 220 and something at 280 also, as I expected.

Nevertheless, your "experiments" do not rule out contamination of the blanks you injected.

If DVB = divinylbenzene then Uwe is getting at bleeding as possible source. I also googled and found this:

http://135.196.210.195/suppdata/CC/b8/b ... 17598e.pdf

This is totally different than the spec above, it has strong absorbance at 220 and something at 280 also, as I expected.

Nevertheless, your "experiments" do not rule out contamination of the blanks you injected.

By "contamination of the blanks" are you referring to mobile phase impurities?

Leaching of the polymeric support is not something I had thought of. If this were happening, would you expect to see single or multiple peaks?

The "dirt" could come rom anywhere. Very often it is introduced by a systematic/consistent carelessness.
In any case, the first thing to do is run a UV spectrum of the peak, it may say something about the source.
On bleeding from polymer columns, Uwe would be more qualified to give an answer.

Bleeding from the polymer is likely to involve a whole slew of complex structures. I do not expect multiple peaks, but one broad peak.