The Drizzled Peak (or, Bumpy Tailing)

[RFRansom]

Heya Y'all;

I have a phenomenon that is puzzling me: the Drizzled Peak.

Here it is:

https://ibb.co/fVXvnkFd

(sorry about the link, I tried selecting it and hitting the Insert image button --- it does flank the link with bracketed img /img, but the post yields broken image icons instead of images)

The lowdown:
Pump = Waters 600 series preparative HPLC pump, part of the old 650e Advanced Protein Purification System (vintage, I know).
Column = 22 x 250 mm Vydac preparative C18 HPLC column
Detector = CombiFlash NextGen 100 MPLC system, output from the HPLC column fed into the inlet for MPLC columns
A = ammonium acetate buffer, B = acetonitrile, flow 25 ml/min, load 2 ml

Yes, it's a rather janky system, but it's been a workhorse for me. I've even used the 650e pump to drive an analytical column at 1 ml/min (it didn't do well with 1 ml/min), and have been doing so since 2021.

I did analytical runs of the various fractions noted in the Drizzled Peak above (color coded):

https://ibb.co/TxTQ7D5z

and this is the analytical run that I based the preparative gradient upon:

https://ibb.co/SDBW1chm

I'm wondering why I didn't just get a peak-shaped peak with a peak-shaped contaminant peak running after it.

Any thoughts?

R

[TylerSmith123]

Lots of questions left unanswered: what are your analytical conditions? Is this a new phenomena or is this the first prep run for this analyte? How does the sample prep look for the analytical and prep sample? Could this be a result of an over-concentrated/loaded sample, what is the maximum concentration and volume you can inject and retain relatively normal peak-shape/chromatography?

These analytical runs that you have posted in particular, do these peaks have an identical RT or is this an adjusted image to make the peaks look nice and congruent?

[RFRansom]

Thank you for your reply, Tyler. I very much appreciate it. You wrote (reply between lines):

> Lots of questions left unanswered: what are your analytical conditions?

22 x 250 mm Vydac 218TP 15-20 µm C18 column
Flow rate = 25 ml/min A = 10 mM ammonium acetate, 8% acetonitrile, pH 5.0, B = acetonitrile, 0.5 ml injected in a 5 ml loop, ~40 mg of compound injected

> Is this a new phenomena or is this the first prep run for this analyte?

Not new. Here's a similar run with another very similar compound:

https://ibb.co/sv4kCZjW

And a run of the same compound but with more loaded (~100 mg in 4 ml), and on a different gradient (NOTE: gradients on the 650e system pump are in increments of 1% B, so the gradient is actually a step gradient as noted in the plots).

https://ibb.co/27Brjnt8

> How does the sample prep look for the analytical and prep sample?

I take the compound down to a crust by rotary evaporation and dissolve in in A, filter, inject.

> Could this be a result of an over-concentrated/loaded sample, what is the maximum concentration and volume you can inject and retain relatively normal peak-shape/chromatography?

I used to run the better part of half a gram of a mixture of several very similar compounds (natural product extract) on the same column using the same pump, and I got relatively normal peak shapes as shown below (some peaks maxed out, it was an analytical detector):

https://ibb.co/ymKS1ZnC

I don't think it's a capacity issue. I've also used the prep pump as pump for a 4.6 x 250 mm analytical column at 2 ml/min with basically no difference between it and a dedicated analytical system in terms of peak shape, though the baseline at 210 nm was bumpy. Which is why I bought the dedicated analytical system, and worry about pump seals and check valves. However, I checked the output of the pump, and the volumes are correct across the %B spectrum.

> These analytical runs that you have posted in particular, do these peaks have an identical RT or is this an adjusted image to make the peaks look nice and congruent?

They have RT within about 0.01 min, I'm quite sure they're effectively superimposable and represent the same compound with various levels of contaminant peaks. The plots are adjusted by the software so that the maximum height peak is of the same height in every plot, obviously from the drizzled peak plot the size of the peaks I clumped together is not to scale, but they are eluting at essentially the same time.

Happy to elaborate or answer other questions, I'd really like to get output from this prep column that resembles the peaks again.

[TylerSmith123]

Hi RF,

I run a lot of Prep and those chromatograms that you have posted look pretty normal. I also have an analytical flow-cell which results in a lot of blown out peaks, but my mixtures are also (seemingly) not as complicated as yours.
So just to simplify this for myself, it seems like your peak is splitting a variety of times that results in this tailing/splitting of one compound with some minor impurities if all of those peaks are the same compound which seems likely from your results. Do you have any standards you could run to determine if this is a column issue versus some sort of sample prep issue? Realistically, a 0.5 ml injection in a 25ml/min flowrate results in a tiny band, and, assuming everything is alright with your injection, would suggest that somewhere along the way your molecules are taking different pathways that results in band broadening. Could it be possible that you have a partial blockage of the column or some frit?
Sorry I can't be more of a help but maybe we can narrow it down furthur.

TS

[RFRansom]

Tyler!

>I run a lot of Prep and those chromatograms that you have posted look pretty normal. I also have an analytical flow-cell which results in a lot of blown out peaks, but my mixtures are also (seemingly) not as complicated as yours.

Yeah, my stuff is natural products, and there are about ten very closely related compounds present, five that I purify commercially. Three are pretty easy, the fourth and fifth co-migrate on C18 reverse phase unless you take particular care with your gradients.

>So just to simplify this for myself, it seems like your peak is splitting a variety of times that results in this tailing/splitting of one compound with some minor impurities if all of those peaks are the same compound which seems likely from your results.

Here's the prep HPLC peak with fractions indicated:

https://ibb.co/fY5RGX1v

And individual analytical HPLC runs, scaled, of the fractions:

https://ibb.co/MDjFxMY3

I really rilly realllly want to get those leading and trailing peaks to resolve away from the main peak. And I don't think its a loading issue.

>Do you have any standards you could run to determine if this is a column issue versus some sort of sample prep issue?

I'm going to try some of the Universal Test Mix that came with my TeledyneIsco MPLC. Should give two peaks on HPLC.

>Realistically, a 0.5 ml injection in a 25ml/min flowrate results in a tiny band, and, assuming everything is alright with your injection, would suggest that somewhere along the way your molecules are taking different pathways that results in band broadening.

Yeah, running 40-100 mg of nearly pure material on that column should give me resolution out the wing-wang.

>Could it be possible that you have a partial blockage of the column or some frit?

It's possible, but the original Vydac C18 I inherited from a lab that was used to death to purify peptides for a core facility never had a guard column, and I ran a veritable poop-pile of relatively crude extracts (I now use an MPLC instead) on that bad boy for years until it finally started losing resolution. The new one is only a couple years old and hasn't been used much, and only for refined samples as a last clean-up step. And it has a guard column.

> Sorry I can't be more of a help but maybe we can narrow it down furthur.

Your spelling of furthur suggests you've read "The Electric Kool-Aid Acid Test".

Tyler, you rock! Great questions so far. I'll run some test mix and post.

[RFRansom]

Well, the test mix did show a bit of dribbling, but not mega. Much less material, but no a negligible amount:

https://ibb.co/3msqh7sv

[Hollow]

Just some thoughts:

- as already mentioned, some blocked frits?
- re-check all the fittings; maybe there is a void somewhere? (even at column head? What about the void time of the column? Is it the same as always?)
- is there some amount of backpressure on the detector? Not getting artefacts from bubbles or siphoning effects
- could you record the pump-pressure? Does it also bump or is it steady? (of course will folow the gradient steps)
- what about some pH issue? What are your molecules? Some kind with multiple nitrogens in it?
- some kind of oligo/poly-desmodic saponins or tannins, so a real mixture of various polymeric variants and quite sensitiv to small changes in solvent composition?
- an (electronic) detector issue? e.g. a lamp going bad with fluctuating emission intensity?
What happens when changing the detector acquicition settings (data rate, filter constants)?

[cigtoxdoc]

If you replace check valves and plunger seals, the problem may go away.

[RFRansom]

Thank you Hollow and Tyler for taking the time. Y'all rock.

From Hollow:

> - as already mentioned, some blocked frits?

Rather doubt it, I've been pretty scrupulous about what goes on it and things like avoiding leaving buffers on or doing the immiscible. Maybe.

> - re-check all the fittings; maybe there is a void somewhere? (even at column head? What about the void time of the column? Is it the same as always?)

Pretty much, and the column is relatively new and has seen only modest use.

> - is there some amount of backpressure on the detector? Not getting artefacts from bubbles or siphoning effects

Again, probably no.

>- could you record the pump-pressure? Does it also bump or is it steady? (of course will folow the gradient steps)

It is NOT steady, bumps up and down like a heartbeat a couple hundred psi. I would love to put in a "new" checkvalve and pump seals on the antique 600-series Waters preparative pump (part of the Waters 650E Advanced Protein Purification System), but good luck finding that sort of thing ~30 years after it was bought. I might have to just load very small quantities.

Thanks again...

[TylerSmith123]

Woah woah woah, the pump is jumping up and down by HUNDREDS of PSI?! I think you found the problem right there. I understand you're running a high flow-rate, but you should expect like 1-2% change in psi when running isocratic. There is definitely something wrong with your pump, and like the commenters above said, likely something disposable. Hell, there are pumps available on Ebay for a thousand dollars that need a once over and disposable replacement that will get you the results you're looking for if you cant find the disposables.

However, if you want a solution right now you may need to go from your binary system into a solitary system with pre-mixed mobile phases and isocratic conditions. Of course, you need to isolate the problem pump (hoping its just one and not both), but the reaper will come for the other pump at some point too-- and much quicker for prep systems. You'll likely need to mess around with some things on your methods (flowrate most obviously), and adjust your injections, but hopefully you'll get some injections as opposed to none at all.

Send us over a picture of this "heartbeat" pulse, if it's what I think it looks like then you're likely dealing with an issue with the pump (and its' disposables).

[TylerSmith123]

Also I found this after a quick google search from a company I have not heard of, it's a performance kit for the Waters 600 pump. I'm not sure if this is succinct for your needs but give it a look if you're able to identify the problem piece: [url]https://www.hplc-asi.com/waters-600-performance-kit/

[RFRansom]

Any recommendations on how to deal with a frit issue? I have a guard column on, should I try one run without it? Or reverse the direction of flow and blow 0-100 and 100-0 a few times? My is 10 mM ammonium acetate pH 5 8% acetonitrile, B =acetonitrile...

[Hollow]

you may also want to have a look at

https://sciencix.com/

It looks like they provide a lot of spare parts for several, even old systems.

most likely a replacement of the plungers and seals and/or the check valves will do the job.

[itspip]

That pump cycling so much really sounds like it needs a good PM. Check for a PM kit that has the seals and possibly the check valves to replace. You could also try to open the purge valve and pull with a syringe adapter to remove any bubbles in the pump/lines from the reservoir to the pump.

[Hollow]

[youtube][/youtube]

Any recommendations on how to deal with a frit issue? I have a guard column on, should I try one run without it? Or reverse the direction of flow and blow 0-100 and 100-0 a few times? My is 10 mM ammonium acetate pH 5 8% acetonitrile, B =acetonitrile...

if you're careful with your samples cleanup and buffers, replace the aqueous one quite often, not toping up the bottles, not rest the system in buffers, flushes the columns at the end of some sequences and even use a guard, maybe the frit issue is indeed unlikely.
I don't even know the porosity for your prep column for the "big" particle size. So I expect it would really need big junk or microbs.

Flushing and storing the column in acetonitrile is ok but then prime the pump with methanol is recommended to keep the check valves happy.

Nevertheless, replacing the guard would be the first step. For a short check, it should be ok to have a blank run without a guard, if the rest of the system is clean, and see if the pressure is steady.
Backflushing of maybe only the guard may be the third option, but the success may vary.