contamination out of water

[ym3142]

Hi, Dear:

I have the following conditions,



Column TSK-GEL Phenyl 5PW RP, 4.6x100x10um,

MP ACN:H2O=20:80 to 55:45 in 25 min Flow 1ml/min

wavelength 230nm



When I have consistently seen one contamination peak at 8.5 min out of no injection(0 ul) or 1ul of (H2O/ACN). This peak has the same retention time as the analyte I am testing so it interferes my test.

LC gave a perfect base line when column was removed off the LC.

After I wash 1) 90% ACN overnight, and 2) 1ML 0.1M NaoH separately, this column gave the same peak in both cases; I tried 4 different of DI water, the same peak there. The peak was present in 30 consecutive injection of 0 ul .



But when I repeated the above condition with a TSK GEL Octadecyl 4PW column 150x4.6x7um, my analyte eluted at 13 min. The contamination peak was also seen at 13 min in the first 3 injections but this peak disappeared in the following 20 injections.

The contamination peak area is about 2k to 5k. Yes, the peak became bigger when the equilibration time was longer.

Question 1, why the two column gave different phenomenon with the same mobile phase? And do you have suggestion to avoid the contamination peak with the 100mm column?

[zokitano]

Hi,

Did you try to replace ACN with new fresh ACN? Maybe your ACN is contaminated. You're using gradient elution where ACN content increases during the run. If it is contaminated greater concentration of impurity will come on the column with time.

Just a thought
Best wishes

[DR]

Different columns, different selectivities, therefore - different retention times.

Search the forums for my posts mentioning Empore extraction disks. That's a pretreatment option for water used in your A phase that should eliminate (or at least reduce) the water impurity.

[Anthony Crawshaw]

We did a lot of work with this same problem using the Empore discs. We had varying levels of sucess and I'm still not convinced.

[tom jupille]

DR is correct, different columns can be expected to give different retention for garbage as well as for analytes

If the Empore filters don't take out the garbage, the other trick you can try is to put a guard cartridge in the line between the "A" pump and the mixer. Of course, this only works if you have a high-pressure-mixing system.

Actually, the best approach is to prevent the garbage from getting in there in the first place. It looks like you have exonerated the water supply (but when you say "4 different" sources, were they all from the same system at different times or from different DI systems? if they all came from the same DI system, I'd suggest getting a bottle of HPLC grade water from one of the usual suppliers and seeing if that looks any different).

The next thing to look at is the ACN (per zokitano's suggestion). If different lots of ACN give the same result, then you will have to dig deeper. Any chance you are seeing residual detergent on freshly-washed glassware? (don't laugh, it's happened!). You didn't say anything about buffers or pH, so I'm assuming you were using straight water. If not, try changing to a different lot(s) of buffer(s), and never, ever put a pH electrode in the mobile phase (to measure pH, pull an aliquot, measure the pH, and discard the aliquot.