Isolation of natural products - help with gradient

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

7 posts Page 1 of 1
I am a undergraduate student working on the bioactivity of certain herbs and plants. I have been trying to further separate my extract using HPLC to test separate compounds for activity, but I have encountered some problems adjusting my gradient.

The chromatogram is shown here: Image. I am using a C18 sunniest column with10.0 mm x 150 mmParticle size 5 µm
The gradient is as such with A: 0.1% TFA Milli Q and B 0.1% TFA Acetonitrile.
Time A (%) B (%)
0 55 45
25 10 90
25.1 0 100
30.0 0 100
30.1 55 45
(Recalibrate for 10 minutes at 45% B)

I'm mainly interested in the 6 peaks labeled in the picture, and I have collected all of them together.
however, I am not sure how to cleanly separate them after. I have so far tried:

(a) A gentler gradient (55%B to 90%B over 25 minutes) but the peaks are slightly broader and still kind of overlapping
=> I am not sure if I am understanding the concept of gradient elution properly, because I presumed that a gentler gradient would have given me better resolution (by increasing k) . Perhaps changing the starting %B is a problem?

(b) isocratic gradient at 30% -> The main peaks (Peak 2 and Peak 5, Peak 6) are nicely separated but analysis time is long and peaks are broad to the point that small peaks (Peak 1,3, 4) cant be detected properly.
Any advice or even a general direction would be greatly truly appreciated.
I would add about 50% Methanol (or even IPA) to your ACN. It is very rare to have a molecule with similar solubilities in 3 solvents. How are you going to determine the bioactivity of each peak? Do you know what each peak is and its concentration?
It a good idea of you to provide a chromatogram, but the picture is rather small...

(a) Going from 45%B -> 90%B over 25 minutes, to 55%B -> 90% over 25 minutes is in my opinion not the best way to test a more 'gentle' gradient. It's only one parameter, but you're changing 2 different conditions in my view (starting condition AND gradient steepness). If you want to test solely a more gentle gradient, increase the time and keep the starting and end %B the same.

(b) If i can see the x-axis correctly, it takes a while for your first peak to elute in that gradient run (which is already long), so I can imagine it takes ages for them to elute at 30% B (or you mean 30% A)?

I suggest to play around (you're learning after all) with different parameters. It's sometimes a good idea to start with big changes; I agree with HPLC chemist that using MeOH is worth a try. You could try a couple of different oven temperatures, columns, flow rates,.. all with a generic gradient. Once you have something that you can work with, continue to fine tune.

Your compounds are rather apolar (flavonoids? carotenoids?) and sit at the head of your column until there's like 50-60% organic, keep this in mind. Maybe using a column with less retention power (C8 phase) gives you more room for playing with the gradient.
as Rndirk said, your compounds of interest elute at around 60%B but within a small range.
So isocratic may be ok but try 55 or 60% instead of 30%
Or try a gradient between 50>70% in e.g. 20 min, followed by a flushing step.
If you can track the peaks in two isocratic runs e.g. 50% and 65%, then you may plot log(k) vs. %B for each peak and interpolate. With that information you may calculate best conditions or see if you need other changes like temperature, solvent or stationary phase as already suggested.

This Waters app-note may also be worth reading for understanding the principles:
"Developing Focused Gradients for Isolation and Purification" ... 2955en.pdf
You might also try a 5mm x 150mm or 5mm x 250mm column and see if you have more separation, provided your system can handle the pressure. Smaller diameter can use lower flow rate and smaller injection volume while giving the same peak height and better separation.
The past is there to guide us into the future, not to dwell in.
If any of your 6 compounds are ionic in nature you can use mixed-mode column for your separation. Mixed-mode columns provide 5-50 fold increase in capacity for ionic compounds, so sometimes you can use analytical column to do your isolation. Right now I am working with some herbal extracts and mixed-mode shows much better selectivity towards this "forest of peaks".
You can do all sorts of gradient and usually double or triple gradient creates very sharp peaks (almost like GC-type)
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
It's easier to tell you what won't work than what will, unfortunately!
(1) Be careful about gentler gradients. Your gradient is already quite slow. What might happen as you make the gradient less steep is that the peaks will indeed get further apart, but they'll also get broader. The consequence is that the resolution may be no better than you have already. It's easy to be hoodwinked this way, and merely makes a method slower.
(2) If you want to go isocratic, you need peaks that are retained, but not too much (retention factor greater than 2, but not greater than 10). I'd agree that 30% is too little organic, giving too great a retention - you've already seen this, and you can estimate it from your gradient. Your peaks elute at 10-13minutes. You probably have a dead volume of about 1 minute on the sort of system you'll be using with 5micron particle columns and 40min runs, which means the peaks eluted between 9 and 12 minutes after the start of the gradient, which began at 45% and increased by a further 45% over 25 minutes. You can calculate from this that the peaks eluted at around 61-67%. Of course this is all very approximate because the peaks don't go from stationary to eluted instantly - but it gives you an impression of the sort of concentration worth trying in isocratic; 60% sounds like a reasonable starting point as suggested by someone else earlier.
(3) Changing solvent: the main benefit of this is that it's easy to do, and costs you nothing. It's worth a try. Don't hold your breath expecting miracles. It might work, but it might just make all the peaks a bit broader and shuffle them round a bit so that they are imperfectly separated in a different way.
(4) Think about your analytes. Do you know what they are? Are there any characteristics they have, that differ between them, that you can exploit? For example, if they have phenolic parts, they may also bind to phenolic columns or perfluorinated phenolic columns. If they have acid or base characteristics, you might be able to get different separations at different pH (but be aware of the pH limits of your column; alkaline-tolerant reverse phase columns are a beautiful thing!).
(5) Size of column: you can improve resolution by using a longer column or smaller particles, but you won't achieve miracles, and a lot depends on your pump. Pressure increases linearly with length of column, but resolution increases with square root, so if your method is running OK at 200 bar and you increase from 150 to 250mm, as well as paying a fortune (10mm diameter columns are expensive) you've now got a pressure of about 340bar and your 400bar pump gets less reliable and the method will be less tolerant of ageing columns and dubious samples - but you'll barely manage a 30% increase in resolution, hardly even noticeable . Smaller particles increase back-pressure massively, so unless you have a lot of spare pump pressure don't even think about it.
In terms of separation, prep LC columns are hideously expensive, so changing column is unfortunately a matter of last resort. Before doing it, you'd probably want to go back to an analytical scale and check if the prospective columns work at a cheaper size before investing heavily. Don't forget, though, that there are lots of other ways to get some purification. Solid phase extraction cartridges are relatively cheap and available in a huge range of chemistries, so you could try pre-purification with contrasting approaches that way.
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