Baseline Issue with Pic Gradient HPLC Run

[RMHPLCGUY]

We're running a gradient HPLC related compounds run with a pic reagent, and having issues with extraneous peaks in the blank shots.

MP A is 0.88 g sodium 1 hexanesulfonate monohydrate, 950 mL 50 mM PO4 buffer and 50 mL CH3CN, pH 3.5

MP B is 0.88 g sodium 1 hexanesulfonate monohydrate, (125 mL 50 mM PO4 buffer + 125 mL water), and 750 mL CH3CN, pH 3.5.

We currently pH the mobile phase by aliquot-the pH probe does not touch the mobile phase. The wavelength is 210 nm and the gradient profile is 0% B at time zero to 100% B at 50 minutes, with 20 minutes reequilibration. The column called for in the method is a Kromasil 250 x 4.6, 5 um. The column heater is set at 35 degrees C.

The standard and sample is dissolved in methanol, and the standard has 6 components. However, our issue is with extraneous peaks in the methanol blank. Even using bottled water and high purity reagents and pH'ing the mobile phase by aliquot, we are getting around 25 peaks or so on each blank shot.

We've tried adjusting how the mobile phase is made, by pH'ing the buffer/pic reagent by aliquot then adding the CH3CN. And we've tried multiple columns. However, none of these things have cleaned up the baseline. It should be noted that most of our peaks are occurring later in the run, roughly 37 minutes onward.

If anyone has any ideas or suggestions, I'm more than ready to read them.

Thanks in advance.

[unmgvar]

can you make your system do a gradient run without injecting the MeOH?
simply run the gradient method and collect the chromatogram?

i would currently guess either impurities from your mobile phases or you have a mixing problem.
can you show chromatograms of those runs?

[AdrianF]

Try diluting the sample in MP A, also try just injecting MP A as a blank.

[tom jupille]

It's not clear whether the extra peaks are coming from your diluent or from your "A" mobile phase (I suspect the latter). The standard diagnostic is to run blank (or even better. "dummy") gradients with differing equilibration times. There's a brief write-up in the LC Troubleshooting Wizard on our web site (http://www.lcresources.com/resources/TSWiz/hs400.htm).

Until you establish the source of the problem, you're shooting in the dark at solutions.

[RMHPLCGUY]

Due to company policy, I am unable to post the chromatograms.

We've done a lot of little things on this run, but nothing helped. The columns we tried were all about the same.

The columns I tried were: the Phemomenex Kromasil, Phenenex Synergi, Phenomened Gemini and Restek Kromasil.

The Kromasils are non polar end capped, as is the Gemini. But the Gemini chrom, while comparable to the Kromasil, was still not substantially better. The Synergi is polar end capped, and was worse than the Kromasils.

Right now, I'm working on the assumption that a good portion of the issues is from the pic reagent and the low wavelength detection. I might try taking it out of both MP's, cleaning my column with MEOH, and shooting a few shots to confirm it.

Also, for the last run we did, we did increase the re-equilibration time to 25 minutes, leading me to think its the pic reagents that are causing the excess baseline noise.

It will be a bit before I investigate further, but I'm going to run the gradient without making an injection, and also try a MP A blank, and also investigate different suppliers Kromasil columns. I'd like to work within the boundaries of the current method, at least initially, before I have to try things that deviate from the method too much.

Thanks for the help.

[seaclimb2001]

I think you are barking up the wrong tree by investigating various columns.

Tell us what brand and grade of PIC reagent are you using? Since you are using 210 nm, it is of utmost importance to use the purest reagent possible. Another source of contaminants is your solvent filter.

[RMHPLCGUY]

Off the shelf EMD 1 hexanesulfonic acid, Na salt, monohydrate.

Ordered the high purity Fluka PIC reagent, but it did not come in prior to the last run.

I'm planning on trying the Fluka reagent next time.

[ebasarah]

Did the peaks come at the same retention time? or it just random?
Recently I encountered ghost peaks problem on my system.

I bypassed the system one by one, and it turned out to be the degasser!

Instead ordering a new one, I used Helium sparging method and I degassed everything before I run my experiment. It solved the problem well