Chromatography Forum

at Luna C18 150x4.6 5u, 20 mM pH2.7 KH2PO4 Buffer, gradient from 10% to 60% MeOH, I found there was a peak coeluting with my propyl paraben after I injected any of water, Buffer, MeOH or ACN.
I tried many different gradient but all the same.
Using pure water in the place of Buffer was the same too.
Is this possible due to impurity out of MeOH?
I already tried to wash column, prepare different new injector wash solution, wash needle injector. None helped.
Is this called system peak?
What is the way to avoid this peak?
Thanks,

I suspect that it is the water. What is your water source and at what wavelength are you detecting your peaks? Run a gradient (no injection) with a long equilibration time followed by another gradient run (no injection) after a much shorter equilibration. If water is the source of the peak, you will see much more of it with the long equilibration.

thanks, I will try.

If you see more of the peak after the longer initial conditions time, you need to clean up the water (search forums for "Empore Extraction Disk").

hi,
you could understand if the guilty is water in a simple way:

you should eluting a mobile phase with a big % of water for a certain period (30' or 1h) and then inject using as eluent your mobile phase (or another with more organic).
If the "peak" results big respect to the previous injections (without conditioning with water) water is the guilty so you could adoperate to find a suitable filtration system....
i'm not sure water is guilty but many times it's...
good luck

thanks, everyone,
the long period of big Buffer running method did work, which gave more/big impurity peaks while under the same method JT Baker HPLC grade water(used to prepare the Buffer) did not gave those impurity peaks.

Glad to hear it, but be aware that even bottled HPLC grade water will sometimes give you these peaks. A C-18 membrane or cartridge filter treatment of contaminated water will usually clean it up.

For anyone who is interested, I finally got around to writing up some diagnostics and fixes for gradient garbage peaks in the "Troubleshooting Wizard" on the LC Resources web site. The specific page is:
http://www.lcresources.com/resources/TSWiz/hs100.htm

We have never needed to use gradient for propyl paraben, and we acidify with acetic acid instead of solid buffer so we don't need to pre-filter the mobile phase or take to a measured pH.

Hi, Consumer Product Guy,
Thank you for your input. But could you please clarify why any of the following related to the impurity issue or any other issues we are discussing here (though I agree propyl paraben give a retention of 5 min if the isocratic mobile phase has 70% ACN for C18. Unfortunately my API retention is 4 min if isocratic mobile phase has ONLY 15% ACN. Between my API and propyl paraben I have two other componets which came at 8 min and 15 min if I run gradient from 15% to 70% ACN for 20 min where propyl came at 20 min. what is your advice?)

"acidify with acetic acid instead of solid buffer so we don't need to pre-filter the mobile phase or take to a measured pH."

DR.
could you provide further information about the c18 membrane and the cartridge treatment of water? any catalog, website, paper? Thanks,

Most labs have a 47mm membrane filtration setup for mobile phase, so we tend to use 47mm extraction disks for water treatment.

Procedure:
First, under vacuum, rinse the disk with a little of your strong (organic) solvent, then water (catching these in something other than your main filtrate collection vessel). Then filter ~1L (more or less depending of the level of contaminants seen) of water, repeat.

Vendor links:
Varian

Fisher

VWR

I'm certain that similar products using different formats are available from other manufacturers and that they work every bit as well as the 47mm disks offered (among other things) on the catalog pages above, but we've found that they are a good match for the fitration setup common in our labs.

Have been looking at similar problems with Acetontrile (see below)

http://www.sepsci.com/chromforum/viewtopic.php?t=4689

My advice would be not to just look at the quality of your water, but also your solvents.

We have been investigating the 3M Empore XDB-XC SPE Disk Filters, with varying degrees of success dependent on the MeCN grade.

Anthony

ACN & MeOH can be qualified for picky gradient methods by simply scanning them in a regular UV/Vis spectrophotometer and picking lots with the lowest cutoff (Best to check these as they arrive, then you can cull the really good bottles before someone else pours them into wash bottles. I think you'll find that they vary considerably).

During my student year we had a problem with bad baselines, eventually solved by getting a new water purifier in. In the meantime my supervisor have me "filter" the mobile phase by pumping it through a column (might have been a bare silica column, it was in my very early days of analysis.)

This could have just been to get me used to working LC's but it did clear up our baseline quite nicely.

It's a bit crude but worth a try if you are really struggling.