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Topic: Confused with the principle of HPLC  (Read 2594 times)

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Offline HJay

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Confused with the principle of HPLC
« on: January 17, 2018, 02:49:27 AM »
Hi, guys

Recently, I had to do a separation of oligonucleotides using reverse-phase HPLC (RP-HPLC) and I was just wondering why RP-HPLC is favored over normal phase HPLC (NP-HPLC). Why can't you just use NP-HPLC to separate oligos? I think the only difference between those two is that their stationary and mobile phase is the opposite of each other. For NP-HPLC, stationary phase is polar and mobile phase is non-polar, and for RP-HPLC, stationary phase is non-polar and mobile phase is polar.

So my question is, since oligos are hydrophilic and polar, can't you just use NP-HPLC to adsorb oligos onto the polar stationary phase and separate them?? What am I missing here?


Offline Babcock_Hall

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Re: Confused with the principle of HPLC
« Reply #1 on: January 17, 2018, 10:17:55 AM »
This is a pretty simple minded explanation.  RP versus NP will "see" (meaning interact with) different portions of an oligo (or a protein).  What the contaminants are relative to the desired material is may determine which type of chromatography is better for any one application.

Offline Arkcon

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Re: Confused with the principle of HPLC
« Reply #2 on: January 17, 2018, 11:32:27 AM »
Again, we have a very simple answer, to a question with multiple qualifiers.

Ahem.

No body does Normal phase HPLC.  It uses awful solvents, is poorly reproducible, and has no advantages against reverse phase.

Now, that statement I just made should come with any of a number of qualifiers.  Yes, normal phase separation is important, even necessary, for some HPLC analysis.  And with proper work, is a perfectly reproducible method, for QC purposes, even cGMP QC analysis.

But reverse phase is so well known, and so easily done, that no one will just flip for the fun of it, as you've described.  That's so far outside of contemporary HPLC analysis, I don't know where to begin to say how its wrong. 

Hey, did you hear, in some nations, people drive on the left instead of the right?  Let's all switch, tomorrow.  It'll be fine.  I'll seed a world-wide tweet tonight.   ;D
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

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