Chromatography Forum

Hi,
Please forgive me for this question, I have only been using HPLC since last Feb. Sadly I am the most knowledgable person in our lab. I am on my own to overcome the learning curve. I have a BA in Biology. The saving grace is we really only test Fermentation Samples. So I just need to wrap my head around that one thing.

Our instrument was set up by the vendor, including methods. Our HPLC is a Waters, pump=1515, autosampler = 717+, detector 2414. The column is IC-Pak Ion Exclusion 7.8x150 mm. What else do you need to know?

After changing the column I have had trouble getting the baseline to look good. The column was shipped in 10% methanol, but our mobile phase is .5 mM H2SO4. I tried allowing the mobile to run at 1 ml/min for a day. This helped some. Today I tried a 5 mM H2SO4 for 30 mins, that seemed to help more. But I am still seeing negative peaks at 7 mins... Always in the same place.

My understanding is that negative peaks are basically a band that transmits more light than the mobile. But that "shouldn't" happen with a mobile blank as the sample.

Questions
1. Can the H2SO4 mobile move the Methanol out effectively?
2. Should I be doing something else to condition the column?
3. How can I have a negative peak when I am running a blank?
4. What causes the negative peak to happen at 7 mins everytime?

Thank you in advance!

My understanding is that negative peaks are basically a band that transmits more light than the mobile

That is true for a UV-absorbance detector, but the Waters 2414 is a refractive index (RI) detector. It measures the amount that a light beam "bends" as it traverses the interface with your solvent flow cell.

Every liquid has a refractive index, and that refractive index can be measured, and the RI will change when things are dissolved in that solvent (as an example, saccharometers are hand-held refractive index measuring devices that are used to measure the amount of sugar in grapes prior to fermentation for wine making). The catch is that while RI peaks in HPLC are usually positive, they can go either way.

So, to answer your questions:

1. Can the H2SO4 mobile move the Methanol out effectively?

Yes

2. Should I be doing something else to condition the column?

No

3. How can I have a negative peak when I am running a blank?

Anything dissolved in a liquid will change its RI (including air). You may be seeing a peak for dissolved air. Try degassing the blank and see if the peak goes away. RI also changes with temperature, so if you you are heating your column and inject an ambient-temperature sample, you will sometimes see a temperature peak.

4. What causes the negative peak to happen at 7 mins everytime?

Because that's how long it takes whatever is causing it to get through the column. You can see an RI peak for every component whose concentration differs between the mobile phase and what you injected. Per my comment above, dissolved air and temperature differences are strong possibilities.

Tom, Thank you for your throrough answers!

Your sugestions seem very likely- Especially the temperature. Our column heating block had been turned off. (no one will fess up )

Also, we have had problems with "sampling" air. One user ran the wrong start up sample set, which called for more vials of standard than were actually in the autosampler.

As you can see our team has a lot to work on. I will look into both sugestions. Thanks Again!