Chromatography Forum

Hi
I am analysing open ocean seawater samples for D and L amino acids using pre-column derivatisaton with N-Iso-L-butyryl-L-cysteine and O-pthaldialdehyde. We're using an Agilent 1100 Series HPLC system with a Thermo Electron Corporation BDS Hypersil C18 column (only been fitted for a month). The solvent program employed uses acetonitrile, methanol, 23 mM sodium acetate buffer (pH 6) and HPLC grade water.
We have all of a sudden lost resolution on the peaks for asparctic and glutamic acid. We have washed the column numerous times, made fresh reagents etc. yet the resolution is still bad. Could this problem be due to the salt in my samples??

How many injections on that column during the month that it's been in service?

About 300 injections, that includes standards, blanks and washes in between samples.

Some packings are incompatible with acetate buffer, have you checked the datasheet of the column for that kind of details ?

I think one thing I would try is to substitute a new column and see if the chromatography returns to normal. That would eliminate all other variables (of which you have a lot of them with this method). If substitution of a new column returns your system to normal, than you can focus on what is going on with your column.

I agree with supercritical, it's the first thing to do....

Definitely swap the column. If it is a column problem, consider using a guard cartridge.

Some columns are incompatible with an acetate buffer????

I am not aware of any silica-based column with such a feature.

Please explain...

For example Novapak phenyl retains acetate buffer and can't be used with citrate buffer (cf doc Waters). But most of packings can be used with those buffer... so i understand your question.

I am absolutely sure that the statement that a Nova-Pak phenyl column retains an acetate buffer is junk. Period!

Citrate is usually not recommended in HPLC since it complexes with metals. It has the reputation to attack steel. On the other hand, I have used it occasionally without trouble.

So if you are so sure you can tell it to Waters..., i didn't invent what is written on the datasheet !, maybe you are right, but they say the oposite, i don't have any opinion about it, and i did'nt use this packing enough to be aware enough.
Regards

Unfortunately, the document is incorrect in several aspects. Just to be clear: the information is not from a Care & Use manual. I'll follow up to get the errors corrected. Thank you for making me aware of this!