Re: Cation exchange column- fronting of cation peak

[CQ_32]

I'm runing the CEX column- hamilton PRP-X200. After 200-300 injections, I saw fronting of my analyte peak. What are the possible factors to cause the fronting? Thank you.

[rhaefe]

You could e-mail a chromatogram stating conditions and a short explanation about the samples and sample matrix to our tech support and have us sort it out...

hplc@hamiltoncompany.com

[HW Mueller]

Some of the rest of us would also like to learn something here. Could it be that the capacity is going "to pot"?

[rhaefe]

Hans,

Of course I can post the out come of our investigation. But we definitely need more info. Change (=loss) in capacity affects retention times. They get shorter. After learning it the hard way I only start my investigations if I can actually look at one (=more are better) representative chromatogram which includes all pertinent separation conditions.

[danko]

Change (=loss) in capacity affects retention times. They get shorter.

Fronting is sort of partial shortening of the retention time. Wouldn’t you agree that when the capacity decreases, there are not enough ion exchange sites which in turn results in faster elution of the abundant molecules, until they interact with some vacant sites, down the column = fronting?

Best Regards

[CQ_32]

the fronting. At first 100 injections or so, we can easily restore the column by multiple injections of 100uL of 1N HNO3, which is recommended by hamilton. However, after 300 injections, even after the regeneration procedure, the retention time of cation still get half of the original one.

I'm wondering that the lifetime of hamilton PRP-X200 column. It seems that the capacity lose permanently. Is there any other way to restore the column?

[rhaefe]

CQ_32:

The fact that the nitric acid injections reverse the tailing/loss of retentions at the beginning indicates that something in your sample, sample matrix is binding to the cation exchange sites on the column. After a while this process is no longer reversible.
What samples are you analyzing and what is the sample matrix?

[CQ_32]

I also conducted the procedure water-MeOH or ACN gradient to remove the protein from the column. The pressure of column didn't increase afterwards. Any recommendation to restore such column?

Thanks.

[rhaefe]

I personally have never tried to remove adsorbed proteins from a PRP-X200 and we don't have a standard procedure for that. What I would try is pumping 10mmol/L nitric acid through the column, preferable against flow direction (back flush). Use a very low flow rate (0.05 to 0.1mL/min) and flush over night.
No guarantee that it will work though.
In the future I would use a guard column and replace it after 75-100 injections or apply the nitric acid injections after every 25 to 50 sample runs.

Since protein adsorption usually happens in the first few mm of the stationary phase you might be able to get a few more miles out of that column by using it against the flow direction.