Chromatography Forum

Dear All

I am using C8 column from agilent technologies to analyse chlorophyll a in sea water samples. The peak shape is now much more broad and looks like column is overlodaed. I have run at least some 1200 samples and now i see this problem.

I use Agilent 1200 series with PDA detector.

Solvent A=70:30 (Methanol:1 M ammonium Ac)
Sovent B=100 % methanol

Column temperature : 60 degree.

Please send me some suggestion

Most probably the column is simply worn out from use. 1200 analyses is not an unreasonable life for a column, and you certainly got your money's worth out of it at around $0.33/injection.

However, there are a couple things to try before tossing it in the trash. First, make sure the rest of the system is OK by putting on a column you know is good and testing it. Next, try cleaning the column with unbuffered 50% MeCN, then acetone, then hexane, acetone again, and 50% MeCN.

If that fails (and you think you have nothing else to lose) open the inlet of the column and examine the packing; a good column will be filled to the top with no gaps or voids. If there is a void, you can empty out a guard cartridge of the same type, make a paste and fill the analytical column. This is a temporary measure to keep you going until the new column arrives.

First, let me congratulate you on running a column at a temperature just a few degrees below the boiling point of your final solvent without problems in the detector.

My only addition to Mark's cleaning would be an extensive wash with highly aqueous mobile phase, possibly at around 40C.

If you have a guard column, are you changing it regularly? - it may protect the column. As he noted, you may have just reached end-of-life, especially at 60C.

Bruce Hamilton

Dear all
Thanks a lot for those suggestion. Last night i had kept it for column flush with 50:50 Meoh:H20.
I do use a gaurd column and may thats one of the reason it had lasted so long at such high temperature.

I will post two different chromatograms of the same sample analysed 4 months back and yesterday.

Oops i feel changing the method will be better option!11

Over 1000 injections is a reasonable life for the column. If you want to improve this, there are a few things to consider.

Did you replace the guard column, and a replacement of the guard column did not imporve the situation?

What is the pH of your ammonium acetate buffer?

The dissolution of the surface of a packing depends on the details of the conditions. If this is the problem, a reduction in temperature will improve the situation.

In future I would suggest monitoring the column efficiency and replacing the guard column when the efficiency reduces by 10% or before each run - this is vey cheap if you pack your own guard columns.

In my 1000 runs i have replaced my guard column twice. Till now i am not quite sure when exactly i should replace it . I did see some black colour deposit. On both the occassion i removed i did not see much changes in resolution until recently when i started seen column overloading signatures.