Chromatography Forum

Hi,

So, I have doing some analysis of some tryptophan metabolites - tryptophan, 5-hydroxytryptophan, n-acetylserotonin, serotonin, and melatonin- on the LCMS. I use a Zorbax 300Extend-C18 column. I noticed a small difference in the separation between serotonin and 5-hydroxytryptophan (they are the first to elute from the column) on a new column. Basically, the old column has better separation between these two compounds than the new one. All the conditions for separation are the same. What could be the reason for this? Thank you.

Are the two columns used 100% exactly the "same"? (1) It is normal to see small differences in separation between "identical"new columns, but this must be properly quantified to see it if is significant. (2) More commonly, differences are observed because the two columns have been treated differently. For example, the surface chemistry of many silica based columns can be permanently changed when ion-pairing agents are used (the column is never the "same" after that and may show different results). (3) The actual method or samples may be different. Small changes in temperature, mobile phase composition, injection solution/volum, detector settings etc can change the results.

Some mobile phase modifiers are notorious for altering column chemistry (things like TEA, for example).
What's in your MP?

Are the two columns used 100% exactly the "same"? (1) It is normal to see small differences in separation between "identical"new columns, but this must be properly quantified to see it if is significant. (2) More commonly, differences are observed because the two columns have been treated differently. For example, the surface chemistry of many silica based columns can be permanently changed when ion-pairing agents are used (the column is never the "same" after that and may show different results). (3) The actual method or samples may be different. Small changes in temperature, mobile phase composition, injection solution/volum, detector settings etc can change the results.

I recently changed the preparation of the standards. I dissolve them in DMSO first, then make up the volume. My first injection on the new column was the standards made in the DMSO. Is it possible that DMSO caused some changes in the surface chemistry of the column?

Some mobile phase modifiers are notorious for altering column chemistry (things like TEA, for example).
What's in your MP?

0.1% formic acid in water and 0.1% formic acid in acetonitrile

Folarin wrote: "I recently changed the preparation of the standards. I dissolve them in DMSO first, then make up the volume. My first injection on the new column was the standards made in the DMSO. Is it possible that DMSO caused some changes in the surface chemistry of the column?"-

Yes, indeed. Changing the prep of samples may certainly change results. DMSO may olny change the surface temporarily, but it is not like water, so best avoided.. Please follow proper HPLC procedure: ALL samples (and stds too) are prepared (dissolved) and injected in the mobile phase solution only (for a gradient, that would be the "initial condition" composition). This insures proper loading into the head of the column. Injection solution choice is very important and should ideally match the mobile phase (and/or be weaker than). This is a basic fundamental. Please work with an experienced chromatographer as the "HPLC" system is just a tool. A tool that requires many years of full-time professional training, with feedback, to learn how to use properly. Acquiring valid data requires an understanding of the technique, instrument and software.