Are LC columns with embedded polar groups better for polar retention? | Trust your Science 4
Articles Blog

Are LC columns with embedded polar groups better for polar retention? | Trust your Science 4

February 27, 2020


– Alright, Kim, what came
in the old inbox today? – Well, okay, let’s see. It looks like we have a question
about using columns with polar embedded groups in them to retain polar compounds. And they’re asking if that is a better option for polar compound retention. – Hmm, well, it kinda makes sense if you think about it, because these embedded
polar columns have an embedded polar functionality
group that’s built into the ligand, so the name implies that they might be good for polars? – I don’t know. I think the best thing
we can do is probably to get a bunch of different columns with polar embedded groups in them, a wide range of hydrophobicities, get some polar compounds,
some non-polar compounds and then run them and see what happens. – Sounds great to me. We’ll fire up some of these
blue boxes and we’ll let it rip. – Okay. – Kim, what we did here to test for this myth is that we used an ACQUITY UPLC H-Class, again, just to test the columns, the four columns that we’re gonna show the data for. We set up the mobile phase solvents with 100% aqueous mobile phase, just again, because polar analytes
like to be solubilized in 100 percent aqueous
and we ran it isocratically, and we’re just going to look
at the overall retention of those phases. – So this is going to show us absolutely whether or not these polar-embedded columns retain polar compounds. – Exactly right, exactly right. – Awesome. – All right Kim, we ran all those columns, results are in, what do you think? – Well, we ran all those columns
with 10 millimolar ammonium formate, CH3, so it’s
100 percent aqueous, that should really show us
what these columns can do. Let’s take a look. – All right, so what we’ve also done here is, we also ran as a standard, a really good reversed-phase polar retentive column, just as kind of a benchmark. I think that was a good thing to do, too, that was the Atlantis T3. And as you can see, when you start looking at all these additional embedded
polar groups, columns, you’ll see that, you know, the retention isn’t quite there, so when you look at the actual different vendors that we looked at here, for embedded polars, you’ll see that some of the compounds aren’t very well retained at all. You’ll see for thymidine-5-mono phosphate, or peak number five for the Atlantis T3, it’s very well retained, or it has very good retention. But when you look at the embedded polar groups, like vendor A, B, and C, you’ll see that peak number five isn’t well retained at all. When you look at peak number four here, adenine, you’ll see that that peak has very poor peak shape, has good retention, but again, the peak shape is just so poor, for an embedded polar group, it’s just not very good. – All right, so it’s pretty clear that the embedded polar groups don’t actually increase polar retention, so why not? How do they work? – Yeah Kim, that’s a great point, I mean, they are really useful phases, especially for reversed-phase chromatography. They offer some additional benefits over straight alcheal chain C18s, and we can look at those now, real quick. So Kim, here’s a nice cartoon of an embedded polar phase, and as you can see, through the chain link there, there’s an embedded carbamate group that’s buried right inside the chain that’s circled. And the functionality of that group, what that does is it allows for some
hydrogen bonding between analytes like phenolic compounds that can add into some differences in selectivity. Which can be useful in chromatography. Also, what’s kind of neat about those phases is that those embedded polar groups will attract water down to the surface, so that’s where the polar kind of comes in, they actually pull water down to that surface, but as you remember from our last episode, being 100 percent aqueous compatible doesn’t give you polar analyte retention. So again, that’s where these myths kind of tie into each other. So if you look at the second cartoon here, what I’m showing is that aqueous layer that kind of builds up on the surface of the particle. And as you can see, what typically ends up happening, is that high aqueous, kind of, shield that goes around the surface decreases or increases the dielectric constant, reducing ionic interactions, especially with ionizable bases. So you get better peak shape for bases. And lastly, what ends up happening is that that functional group, that carbamate group, can kind of bend back on itself, and protect itself over unwanted silanol interactions. Again, creating better peak shape for bases. So Kim, based on the information that we showed today, I think it’s clear to see that embedded polar groups are really important in reversed-phase chromatography, they’re just not very useful for polar compounds. – Yep, I think we can safely call this myth busted. – Busted for sure. – Let’s go ahead and type up our response to this scientist and help him out. – Great. – If you’d like your question to be answered on a future episode, please feel free to email us at trustyourscience at waters.com

Leave a Reply

Your email address will not be published. Required fields are marked *