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Chemistry Forums for Students => Analytical Chemistry Forum => Topic started by: gumusonur on March 16, 2022, 08:39:47 AM

Title: hplc speration
Post by: gumusonur on March 16, 2022, 08:39:47 AM

we work on aminoacid in HPLC-DAD.
The following two molecules are similar to each other, only their molecular weights are different. How should I go about separating them? Which parameters should I change?

Title: Re: hplc speration
Post by: Babcock_Hall on March 16, 2022, 02:00:44 PM
What have you thought of so far?  Also, when this question was asked, was the stationary phase specified?
Title: Re: hplc speration
Post by: rjb on March 16, 2022, 04:18:59 PM
If you give us as much information as you have about run conditions that would certainly make things easier.
Are you carrying out derivatization? I presume so, but if not that is certainly something that you will want to consider and is pretty much par for the course for AA analysis. OPA and FMOC seem to be the most commonly used agents and will help to increase retention times (I presume that you're carrying out RP-HPLC with an appropriate column?) and improve separation and will increase S/N and hence sensitivity.

Title: Re: hplc speration
Post by: gumusonur on March 18, 2022, 05:21:21 AM
Yes, I am derivatizing. you can see method conditions.
column temp: 43 degree, sample temp: 20 degree
Title: Re: hplc speration
Post by: rjb on March 19, 2022, 05:13:21 AM
If my inferences are right then I suspect that the solution to your problem lies with somebody with very specific expertise in the method and chemistry that you are using... Probably not this forum.

Judging by the image you provided, your HPLC has a quaternary pump and I don't recognise the interface, so it's probably a Waters system? A very nice and rather capable one by the looks of the parameters!

I wondered why you didn't specify the column or specific mobile phases, so I assume that either you don't want us to know (which I thought was unlikely as it's hardy likely to be part of an NDA) or perhaps you don't know. If the latter then you're probably using a proprietary kit like AccQ-Tag which includes 2 eluents which contain who knows what, although the MSDS might give you some idea.

If this is right, then to be quite honest, your best option would be to talk to Waters who should be able to provide some advice. You might also like to look up a publication by somebody called Fosia Dahir Jada from the University of Uppsala if you haven't already.