How to Separate Coeluting Compounds

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I am running an HPLC -UVvis assay using methanol and water (88% to 12%) and the retention time of my two analytes are 13 and 14minutes and are coeluting using my reversed phase C18 column.

The method is an isocratic method.

Does anybody have any ideas how to better separate these compounds?
I was thinking to evaluate Acetonitrile instead of Methanol to check the selectivity, as well as to evaluate a gradient, but these compounds seem to be already pretty strongly retained at very high organic. Any idea of what gradient to use, or mobile phase suggestions?

I would Kindly appreciate any tips.

Best Regards.
First of all, if the retention times are different, they may not be fully resolved but they are not coeluting.

Without more detail, it's hard to be specific. What dimensions of column? What flow rate? What particle size?

Since you can measure the retention times, I presume you have run individual standards. What are the peak widths?

The peak widths and retention times will give the plate number. Knowing column dimensions and particle size will tell you whether that plate number is reasonable. If it's not, then you need to fix the problem. If it is, then addressing selectivity is next. 88% methanol is not super-high, and acetonitrile is generally a bit stronger than methanol, so it takes less of it to do the same job, which means that switching to acetonitrile is certainly feasible.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Im using a Luna C18 column with dimensions of 250mm x 4.6 i.d. and a 5um particle size. The flow rate is 1.0mL/min.

Thanks again.
By the way, you're using a decent statioanry phase of relatively old-school dimensions and particle size...

OK, inject less volume of your standard, see if the peak shape/resolution improves.

Try ACN, maybe at 70%, but don't be surprised if things don't change much.

Feed in a little THF, start at 10% while decreasing the level of the ACN/methanol.

Try gradient elution with your aqueous and either methanol or ACN.

Try different C18 phase or a different phase.

Your retention times are a minute apart, so you don't have to start from scratch here, this is the fun stuff.
I'm with Tom and the others who have responded:

1) Make sure your column is performing well (measure theoretical plates)
2) Back off the MeOH some (or better yet, use MeCN), and..
3) Elevate the column oven temperature
If you can, try a different stationary phase, like a phenyl column. You can also try to determine if pH has an influence on retention - try a phosphate buffer at pH 2 or acetate buffer at pH 4.8.
Time flies like an arrow. Fruit flies like a banana.
DJ wrote:

3) Elevate the column oven temperature



Yeah, try cooler and warmer column temperatures. For both: ask whether separation is better, worse, or the same, and use that hint.
Indeed we tried 88:12 ACN:Water, the resolution seems slightly better but one of the actives does not show up even for a 50ug standard. Also using 2 and 5% THF did not work so will try 10% and more Also thinking to try adding 1% acetic acid. Might have to probably evaluate another column.

These are picked up at 302 and 305nm max absorbance and very similar in structure.
With all due respect, it looks like you're "flailing": trying things at random and hoping that something will work.

My $0.02 worth would be to do it systematically:

1. How much resolution do you need? 2.0? 1.5? FWIW, the FDA "suggests" that Rs should be > 2, but you may need more if there is a big disparity in your peak sizes or if there is a lot of tailing.

2. The classic relationship for what controls resolution is Rs ≈ (0.25) * SQRT(N) * (alpha -1) * k'/(1+k')

3. Your column is 250 x 4.6mm, which means that it has a dead volume of around 2.5 mL. At 1 mL/min flow rate, that's a dead time of 2.5 minutes. Retention times of 14 and 13 minutes then work out to k' values of 4.6 and 4.2, respectively, and that, in turn means an alpha value of 1.1. You didn't say how wide your peaks are, so I can't estimate the plate number, but if we assume N = 5,000 (which would be mediocre for a 250 mm column packed with 5 micron material), that works out to a resolution of about 1.4.

4. So you have three possible approaches to improving resolution:
- increase k'
- increase alpha
- increase N

5. If you increase k' from 4.4 (your current average) to 10, that should increase Rs to about 1.6 (assuming nothing else changes).

6. If you can increase the plate number from 5,000 to 10,000 (you should be able to get 10k plates on that column geometry) that should increase Rs to about 1.9 (again, assuming nothing else changes).

7. alpha (selectivity) is where you have the most leverage.

In reversed-phase, there are only six things you can do to change alpha:
- change the % organic (which changes k', but can also sometimes change alpha)
- change the temperature
- change the organic solvent
- change the pH
- change the column
- put in additives (e.g., ion pair reagents).
So now you can be systematic, as suggested by the preceding posts. Take each of those parameters and try tweaking them.

8.
- %B. Change it by 5% (either way). Did the resolution get better or worse? If it got worse, go 5% the other way and see what happens.
- temperature. Increase it by 10 degrees. Did resolution get better or worse. If it got better, increase is some more. If it got worse, decrease it.
- organic solvent. Switch from methanol to acetonitrile, then tweak the acetonitrile 5% either way to see if selectivity changes. If you don't object to THF, repeat with that (a lot of labs don't like THF in part because of the high UV cutoff, but that shouldn't be an issue in your case).
- pH. Are your compounds acids or bases? If so, what are their pKa values (if you don't know exactly, estimate from the structure and functional groups). Start 2 pH units away from the pKa and then run a series with decreasing (or increasing) pH moving toward the pKa in half-unit intervals. [Since you were using plain water initially, my guess is that pH won't be very effective].
- column chemistry. Rather than guessing, go to the USP column selectivity database and look for the most different selectivity:
http://www.usp.org/app/USPNF/columnsDB.html (there are actually two databases there; the second one (PQRI) has a lot more columns). Then start over from the top with the new column.
- additives. Same general comment as pH; these are most effective when you have acids or bases.

What this basically amounts to is a "QbD" (Quality by Design) approach to developing your separation. Although this seems more time consuming than guessing right the first time, it pays off when you go to validate the method, because you have a lot of data about robustness with respect to the various parameters.

Sorry for the long-winded post! :roll:
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Thank you all very much for your input!
Do I dare to offer mixed-mode solution? If your compound is ionizable and hydrophobic you can do it with mixed-mode chromatography. Here is what happening when RP or IE fail:
http://www.sielc.com/Technology_2D_Properties.html

Look trough applications, may you will find something similar:
http://www.sielc.com/Applications_By_Compound.html

Contact me if you have questions. We are offering free method development screening.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
tom jupille wrote:
1. How much resolution do you need? 2.0? 1.5? FWIW, the FDA "suggests" that Rs should be > 2


My guess is that the FDA has guideline of two in a validation, figuring that it won't get better as the column ages.

I had already mentioned that his column dimensions, particles size are so "yesterday".
Tom's absolutely right.
(1) If you have enough retention, then merely increasing it won't improve separation noticeably.
(2) Unless your column has failed catastrophically, increasing column-length or decreasing particle-size with a view to improving peak-width only gives smallish benefits. For example, doubling the length of the column doubles the back-pressure and improves resolution by square-root-of-2 at best (at best, because you may end up forced to choose a less suitable flow-rate because the back pressure is otherwise too high).

If you are lucky enough to have access to a completely different column, then try it. I agree with bisnettrj2, phenyl is a useful alternative choice. It's a bit like pressing the "hyperspace" button in the asteroids video game: it's not a particularly logical or orderly thing to do, but it will suddenly give you a completely different profile, different things will coelute, and if you're lucky, your compounds of interest will not. I definitely think it's more useful to go for a wildly different column than merely tinker with other C18 phases which are all fairly similar. If you do feel an alternative C18 is enough, then pick one which is very different to your C18.

Of course the best thing is to look at your two compounds and see if any bit of them is exploitably different. They obviously have similar hydrophobicities, but if one has an ionisable group that's missing in the other, then an appropriate pH might differentiate the two. If one is more aromatic, a phenyl column is a good choice.

Changing to a more modern column dimension won't help your problem at all, but it's a good thing to do generally. You could get a very similar quality of separation from a 2mm *100mm Luna column with 3 micron particles, run at about 250uL/min (there are calculators that will make the comparison properly; I'm plucking figures from mid air) which would reduce your solvent consumption 4-fold, and still be totally compatible with almost any LC system. If you're feeling more adventurous you could even move on from Luna (which is still a very good packing) to something like Kinetex (solid core; separates like a smaller particle, back pressure like a bigger one).
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