I am having problems with ghost peaks and cannot seem to pin-point where they are coming from. My HPLC system is Waters 515pump,717injector,486UV detector and 746integrator. Mobile phase is 0.01N H2SO4 and the column is a USP L17. I am developing a method to detect gluconic acid in rinse solutions. The ghost peaks are two to three closely eluting peaks at 2mins and one very reproducible peak (area and RT) at 11mins. The peaks do not interfere with the gluconic acid peak (5min) but I need to explain where these peaks come from. I have recently installed a new lamp in the detector and the energy is optimal, also cleaned the flow cell and injector as per Waters instructions and the problem persists.I then tried to perform the run on a second HPLC system exactly the same make/model of the first system and the ghosts peaks also occur. Additional conditions are: Injection vol. 40 uL, UV at 210nm, 0.100AUFS
Thank you for any suggestions.

I don't have a copy of the USP at hand, what is an "L17"? From the mobile phase, I suspect it might be an "ion exclusion" column. If so, you might be looking at the "total exclusion" volume for the first peak (i.e., the elution of fully charged species which are excluded from the interior of the resin beads and so elute in approximately the interstitial volume of the column) and the "total permeation" volume for the last peak (i.e., the elution of neutral species which do not stick to the resin but can access the total liquid volume of the column). The ratios of the retention times for your "ghosts" sound like they're about right for this explanation.

You can check for the former by injecting a dilute samaple of a UV absorbing strong acid anion (nitric acid seems like a reasonable choice). I'm not sure about a good probe for the total permeation volume. Formaldehyde might work. If I were doing it, I would run formaldehyde, acetaldehyde, and propionaldehyde and extrapolate back to zero chain length, but this is probably overkill for your purposes.

If your column is *not* an anion exclusion column, then disregard the preceding drivel!

-- Tom Jupille / LC Resources Inc.

Thanks Tom for your response. The description of the L17 column in the USP is a "strong cation exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the hydrogen form". The manufacturer's literature has this description, also adding that the use of the column is for "analysis of monosaccharides in combination with organic acids, alcohols, ketones, fatty acids, or other neutral compounds". The column is 300mm x 7.8mm and the samples are prepared in purified water. From what I could gather, these ghost peaks are characteristics when using this type of column.

Our major concern is that these ghost peaks appear even when I inject mobile phase or purifed water. A zero microliter injection reveals a clean baseline.

We discarded the posibility of system contamination because, we have the same behavior with another similar system and also after changing the "Seal pack" and syringe of the instrument. The instrument performance was also verified by qualified personnel.

We think that maybe there is a posibility of dissolved gases in the liquid injected, because, if we purge the liquid prior to injection, the 11 minute peak area decreases sustantially and the 2 minute peak does not change significaly.

If you would like to talk with us, we can be reached at (787) 793-7288. This is a direct line, and my name is David Ramos. Thanks again.

OK, that is the "ion exclusion" column that I was thinking of. Those were developed in the late 70's by Larry Cummings, who's on the advisory panel here (Bio Rad HPX-87H). He may be able to comment further.

From here, it looks like you've pretty much confirmed that the 11-min peak is dissolved air (oxygen). Oxygen *does* absorb at low wavelength. See the "LC Troubleshooting" column in the April, 1998 LC/GC for a good example [LC/GC, 15(4), 328 (1988)] if you need to convince someone that you're not making it up.

The 2-min peak(s) still sound to me like the "total exclusion" peaks. If you think of this as resulting from the upset in column equilibrium caused by the injection, it makes it easier to accept. I wouldn't lose any sleep over it. If it helps, referring to them as "system peaks" is much more reassuring than calling them "ghost peaks" ;-)

-- Tom Jupille / LC Resources Inc.