Chromatography Forum

Greetings everyone. I desperately need your help. My gradient's mobile phase consists of 0,2% H3PO4 and ACN. The total run is 20 min. From 15 min to 17 min I included an isocratic step of 95% ACN. And here's the problem: ghost peaks are appeared at the retention times of my analytes. The wavelenght of the analytes absor ption is 210 nm, the same that the ghost peaks appears. How can I make the dissapear?? Please help.

There are so many possible options for ghost peaks its difficult to help without a lot more info and the results of a number of tests but this Agilent guide is useful for working through the most common causes

https://www.agilent.com/cs/library/data ... _notes.pdf

good luck,
Chromavore

Try to make a shallow gradient (5-20 min) instead of the steep one (only 2 min) in order to separate the analyte peak and the peak resulting from the gradient.

Search this forum for Empore Extraction disks.
Do the testing suggested (find out if longer stints at initial conditions lead to larger ghost peaks) to see if the peaks are coming from your A phase's water. If it is, use the EEDisks to filter that water before making your A phase.

In addition to the Agilent Guide and DR's post
Have a look at the Troubleshooting Wizard, #8 ExtraPeaks
https://www.lcresources.com/resources/TSWiz/hs100.htm

The Empore Disks are also mentioned there. Or filter your aqueous mobile phase through a C18 SPE or flash column.
Maybe also give your system a cleaning and maybe replace your reservoir inlet frits.

We run a similar gradient on some of our LC methods at 214nm with the same mobile phase. At the start of 2023 we started seeing ghost peaks near the top of the gradient (90% ACN for our iso step). There were 2-3 consistent ghost peaks that would appear near the top of the ramp and early on in the isocratic step. After many many different trials we finally traced this back to bad Acetonitrile.

At that time we were purchasing VWR branded ACN (their supposed HiPerSolv CHROMANORM grade). It was so bad with some batches that it was completely unusable. After some back and forth with VWR, we discovered they had recently changed suppliers for the chromatography grade ACN. However, they continued to attest that it met all of their testing specifications for chroma grade ACN. So we started shopping around and purchased a sample of 6 or 7 different brands of chromatography grade ACN. We found the same impurities in many of those purchased. It seems that most of these other brands we tried are just re-selling the same ACN that VWR is selling...figures. I dont know how many acetonitrile producers are in the world, but many brands seem to be purchasing from the same supplier. We finally settled on a grossly more expensive Fisher "Optima" grade ACN, and we have had no issues since.

So we started shopping around and purchased a sample of 6 or 7 different brands of chromatography grade ACN. We found the same impurities in many of those purchased. It seems that most of these other brands we tried are just re-selling the same ACN that VWR is selling...figures. I dont know how many acetonitrile producers are in the world, but many brands seem to be purchasing from the same supplier.

Very few actual ACN manufacturers. Remember the "ACN shortage" that began in 2008? Apparently ACN production was a by-product (2-4%) of the manufacturing of acrylonitrile using the Sohio process so ALL suppliers were short/out; vendors had been just selling same stuff, different labels. See https://www.chromatographyonline.com/vi ... or-live-it

So some suppliers rationed supply to a few "regular customers". I purchased a case or two of ACN that already contained acetic acid, as we were adding acetic acid in our isocratic cGMP separations anyway, and could achieve the exact same mobile phase composition that way. We had enough of that and 100% ACN to get us through at least half a year at R&D, and our manufacturing/QC also had enough.

Anyway/of course my pointy-haired boss figured that ACN would be unavailable forever, so he had us develop/re-validate cGMP methods using an alternate solvent so we did (methanol). Of course methanol was less expensive (but also is bad environmentally like ACN) but like many other chromatographers we had used ACN because system pressures are lower than with methanol. Anyway, we did all that development/validation work, turned out to just be a learning process as ACN did become available before we ran out.