GLYCINE Problem Related Substances

[crimelist]

Hi.. I have a problem with resolution of Glycine and Impurty A.

This is the EU Method.

I changed 3 different columns.

Any idea??
Thnks!

[HPLCaddict]

A bit more of information would be nice...

Did you try the column specified in the knowledge database as suitable (Purospher Star RP18) or some other kind of "equivalent" column? If you used a different column it obviously is not equivalent for this application and the easiest thing to do would be to get the Purospher.

If you indeed used the specified Purospher Star column and resolution is not sufficient, we'd need to look a bit deeper into the chromatography.
Resolution is a function of selectivity, efficiency (aka plate count) and retention factors. Compare your chromatogram with the exemplary chromatogram available at the knowledge database. Where are the differences? In absolute retention times (-> changed retention factors), in relative retention times (-> changed selectivity) or in peak widths and/or peak shapes (-> bad efficiency)?

[crimelist]

A bit more of information would be nice...

Did you try the column specified in the knowledge database as suitable (Purospher Star RP18) or some other kind of "equivalent" column? If you used a different column it obviously is not equivalent for this application and the easiest thing to do would be to get the Purospher.

If you indeed used the specified Purospher Star column and resolution is not sufficient, we'd need to look a bit deeper into the chromatography.
Resolution is a function of selectivity, efficiency (aka plate count) and retention factors. Compare your chromatogram with the exemplary chromatogram available at the knowledge database. Where are the differences? In absolute retention times (-> changed retention factors), in relative retention times (-> changed selectivity) or in peak widths and/or peak shapes (-> bad efficiency)?

Thank you. I used two columns Luna C18 and 1 similar column.
Unfortunately I do not have a reference chromatographic profile . This is the first analysis.
I also changed pH ( raising it and lowering it ) . I got max 1.8

[HPLCaddict]

The EDQM knowledge base is accesible free of charge. Details for the glycine monograph can be seen here:
https://extranet.edqm.eu/4DLink1/4DCGI/ ... w/mono/614

There is also a chromatogram (just click on the "availabe" next to "chromatogram").

The first thing that hits the eye is that in your chromatograms the peaks are too early! The first peak elutes at ~2.5 minutes, which corresponds roughly to the dead time of the method (for a 250x4.6mm column at 1mL/min). This means you don't have enough retention. In the exemplary chromatogram, the first peak is at ~4 minutes (which still means very low retention with k~0.6, but at least there is some retention).
Furthermore your peaks don't look very nice concerning asymmetry and width, but the lacking retention is the first thing to care about. Check and double-check that the mobile phase has been prepared correctly, than prepare a new batch of mobile phase and try again. The method uses an ion-pairing reagent, so the column needs some time to get fully equillibrated - to be sure, you may equillibrate the column overnight at a low flow rate.
If this doesn't help to get retention, get hold of a Purospher Star and try that column.

[HPLCaddict]

Looking at the method again, a further thought came to my mind: This method uses a purely aqueous mobile phase. This is something you should NOT do with a usual hydrophobic C18 or you might get phase dewetting / phase collaps. Luna C18 is definitely a hydrophobic column which is not suited for such a mobile phase. Maybe that's the cause of the problem?
I'm not sure about the Purospher Star, but it might be better suited for purely aqueous mobile phases...

[Kreall]

The problem is obvious. The Luna column can not withstand 100% aqua mobile phase. The Merck Purospher Star can.
https://www.merckmillipore.com/PL/pl/pr ... V3.Lxi,nav

You need to get a column that withstand a mobile phase containing 100% water and that will solve your problem.

[crimelist]

The problem is obvious. The Luna column can not withstand 100% aqua mobile phase. The Merck Purospher Star can.
https://www.merckmillipore.com/PL/pl/pr ... V3.Lxi,nav

You need to get a column that withstand a mobile phase containing 100% water and that will solve your problem.

Thanks... now I try!!!

[crimelist]

The problem is obvious. The Luna column can not withstand 100% aqua mobile phase. The Merck Purospher Star can.
https://www.merckmillipore.com/PL/pl/pr ... V3.Lxi,nav

You need to get a column that withstand a mobile phase containing 100% water and that will solve your problem.

Thanks... now I try!!!

Nothing... Resolution 3!

[Kreall]

you changed your resolution from 1.8 to 3 so that is not nothing.
What column did you use?

[crimelist]

Yes yes.. right!


Thi is my new result:



The columns is: Purospher® STAR RP-18