HILIC: Issues with Spermine/Spermidine carryover

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

13 posts Page 1 of 1
Dear all,

I looked for someone to have posted a similar topic but I couldn't find any using the search function.

My issue is as follows:

I am working on a method for amine analysis by HILIC-MS/MS. Two of my analytes are spermidine and spermine. These two analytes arequite tough to get nice peak shapes, so that i have adjusted my conditions accordingly. Unfortunately, even with those conditions I can see those compounds carrying over into the next runs. I have confirmed that it is those analytes in the following runs by retention time and ms/ms.

First off, conditions are as follows:
Eluent A: 90% ACN, 10% H2O, 100 mM ammonium formate, 4.5% formic acid, pH 2.5
Eluent B: 100% H2O, 100 mM ammonium formate, 4,5% formic acid

0->3 min: 100% A
3->7.5 min: ramp to 42% A
7.5->8 min: hold at 42% A
8->10 min: ramp to 100% A
10->12 min: hold at 100% A

Flow: 0.5 ml/min

columns: I have tested this method on both an Agilent Poroshell 120 HILIC-Z (10 cm) and a Thermo Fisher Accucore HILIC (10 cm). Both columns show the same carryover issues.

I also want to provide some further explanation of the issue. English is not my mothertongue, so please excuse any wording and logic errors.
After injecting a sample containing spermidine and spermine, I will detect a fraction (~10%) of the sample response in every further run (even water and blanks) I do. I first realised this was an issue already a few hundred runs into method development. By now I have carryover in every run, so that I always detect a small amount of spermidine and spermine. It isn't as much as I will inject with any sample containing the analytes but it is enough to change the quantitative results significantly.

I have already tried extensive troubleshooting by excluding certain parts of the LC for the same blanks. Thus, I can exclude the Eluent Pump, sample manager and MS as root cause. I am almost 100% certain, that the reason for carryover lies within the capillaries leading to and the column itself.

I have already tried cleaning those with varying cleaning solutions used as eluent:
pure water, water:IPA 50:50, ammonia pH10, ammonia pH11, acetic acid pH2.5.
None of them have significantly lowered the amount of carryover, even though i have done 10 runs with every cleaning solution.

I am very grateful for any input and ideas on how to deal with this issue.

Thank you in advance,

Dominik
You don't say what solvent your sample is in when injected. It's relevant.

The carryover problem is probably caused by cation-exchange of these polyamines with the stationary phase. "Zwitterionic" ligands are supposed to be neutral, but they actually act like cation-exchange materials of reasonably high capacity under some conditions. Let's assume that's the case with your HILIC-Z column. You didn't say which Accucore HILIC column you're using; there's more than one type. In any case, all silica-based "neutral" coatings exhibit some degree of cation-exchange properties.

If polyelectrolytes like these have different counterions, then the resulting ion pairs will differ in polarity and will migrate through the column at different rates. That can account for broad peaks in HILIC. A solution is to make sure that all of the ionizable groups in the sample have the same counterion. Your use of 100 mM ammonium formate is a pretty good way to do that, but you might find that 200 mM would be even better [again, what salts are in the sample solvent?]. This should lessen the degree of carryover as well.

An alternative approach would be to use an anion-exchange column in the HILIC mode. The column surface would repel the polyamines electrostatically, but the hydrophilic interactions will force them to associate with the column anyway. This combination is called ERLIC. The 90% ACN that you're starting with should be enough to guarantee retention. The repulsion should preclude any carryover.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
Dear Andy,

thank you for your swift and helpful reply. I will definitely try 200 mM ammonium formate and keep you posted on the results.

Sorry for not providing information on the sample solvent. I have tried different approaches already:
Water, 1% formic acid and eluent A. I have found eluent A to produce the best results in terms of peak shape. It made no difference, however, regarding carryover.

The Accucore HILIC column is, as far as I know, just called Accucore HILIC. It is the 100x2.1 mm version with a 2.6 µm particle size. The Thermo Fisher part no. is 17526-102130.

Dominik
Hi Dom,

Something that may be an issue here is the column's lifetime. It seems like originally this issue was not happening for you, but has started to develop over the course of hundreds of runs/injections. Do you know roughly the total number of runs on this column? Andy brings up a great point about the cation-exchange of the zwitterionic ligands and columns. I have a feeling that your column may be dying.

I am not an expert in the column that you're talking about, however the mobile phase you are using is particularly harsh with almost 5% formic acid. This could potentially contribute to some overall lifetime issues on your columns. Either way, it seems like your analytes are sticking to your mobile-phase too well. But you should detect this on your injections if you have a calibration curve set-up for your injections and this should be your largest indication that the column is holding your analytes and needs to be washed. There are a handful of great cleaning procedures out there, I would just look one up or look in the Agilent or Thermo-Fischer's documents for their's and perform it on both of your columns.

TS
That particular Accucore column is uncoated silica. Silica can be used for HILIC, but it's also notorious for its negative charge due to ionization of the silanols. I daresay that if you used a column with a neutral coating for HILIC, then you could get away with a much lower percentage of formic acid in your mobile phases. As things stand now, neither of your columns is particularly good for analysis of polyamines.

Peak shape: If there's a significant mismatch (> 10%) between the amount of organic solvent in the sample and the amount in the starting mobile phase, then the injected sample can migrate through the column without mixing at all with the mobile phase (result: Elution in the void volume) or mixing via streamers that are torn off the edges of the sample plug and move downstream faster than the rest of the sample. Result: Broad or ugly peak shapes. That explains why your use of Mobile Phase A produced the best peak shapes.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Hi Tyler and Andy,

thanks for your replies.

@Tyler:
I do know the amount of runs on the columns exactly, as I have already tried using new columns after detecting this issue on an old column with many runs. With these new columns i have instantly had the same issues.

I am aware, that the conditions are quite harsh for the column. However, other methods are running on the same column and with no adverse effects.

I am not quite sure if I get your last point. If the analytes are sticking well to the mobile phase, why aren't they coming off the column? I always thought they stick to the column. I have already tried some cleaning procedures with no significant success.

@Andy:
Thank you for your input on the column type. In my opinion, the amount of formic acid is mainly required for homogenisation of the eluent A (as 100 mM ammonium formiate isn't miscible with ACN in 10:90 without a high concentration of formic acid).
Furthermore I would like to know if you have an idea for which type of neutral coating you would recommend? Maybe you even have a certain column in mind?

Thank you in advance!

Best,

Dominik
You could increase the solubility of the salt by using triethylammonium formate instead of ammonium formate.

HILIC columns you may wish to consider that have neutral coatings:
1) PolyLC's own lovely PolyHYDROXYETHYL A. This is available in packed capillaries as well as columns, in case you want to skip the flow splitting prior to the MS/MS analysis.
2) Advanced Materials Technology's HALO Penta-HILIC columns.

I'd avoid Tosoh's Amide-80; it's quite acidic.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Dear Andy,

thanks for your advice on the column and the salt.

I have taken triethylammonium formate into consideration before. Unfortunately, this isn't practicable, as one of the compounds I am trying to analyse with this specific method is triethylamine.

I wanted to keep you posted on the success of using 200 mM ammonium formate: the overall carryover in the following runs has decreased by ~20-25% (from ~150,000 counts to ~115,000 counts of area) when compared with a salt concentration of 100 mM. I will try some more runs without injections in order to see, if carryover reduces and possible gets cleaned out over further runs.

Thank you for your input on column choice. I will do some more research on that!

Furthermore I will also try using ERLIC, as we happen to have a suitable anion exchange column by hand. Updates will follow.

Best,

Dominik
Instead of triethylammonium formate, you could use either tetramethylammonium formate or diisopropylethylammonium formate. You would just add the relevant amine to a solution of formic acid to get the salt at the desired pH.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Hi Dominik

First, I don't have hands-on experience with these kind of analytes. So I just want to throw some thoughts on you issue so far.

May we ask for the LC system that you're using? Maybe certain devices call for special care and procedures.

I have confirmed that it is those analytes in the following runs by retention time and ms/ms.

Thus, I can exclude the Eluent Pump, sample manager and MS as root cause. I am almost 100% certain, that the reason for carryover lies within the capillaries leading to and the column itself.


Then the other posts are mainly focusing on the column and your mobile phase.

So I want to clarify if you're speaking of real carry-over, e.g. real but smaller peaks at the exact retention time of the substance, or erratic occurence at random retention times or noise background with the molecular ions?

For my logic, if the retention times are the same as in "normal" injection, then the carry-over part has to stick somewhere in the injection device. Because, the other parts of the flow path (tubing, column) are constantly washed with mobile phase actually with high elution strength at the end of your gradient. Therefore, if the substance is sticking to the tubing or column, I would expect more of a wash-out with noisy baseline or peaks at random retention times towards the end of the gradient.

If the retention time is reproducible and the same as normal, then the carry-over fraction behave just like a small sample (is "online" spiking your injection). Which then has to occur somewhere in the autosampler path (needle, needle seat, injection port (Rheodyne valve?), loop).

How did you exclude the autosampler? What troubleshooting have you done for this?

Maybe try to start the injection without involving the autosampler (in Empower "inject immediate"). Or construct a gradient that mimmic multiple injections by just repeating the gradient program at its end.
e.g. (2 cycles)

Code: Select all

min   %A
0   100
3   100
7.5   42
8   42
10   100
12   100
15   100
19.5   42
20   42
22   100
24   100
etc.


If you still observe the carry-over in the second and later cylce, then your analytes are sticking somewhere just in front of the column (would be weird but who knows).

If there is no carry over, then the autosampler is back in the game, I guess, and the column isn't to blame.

Have you changed your needle-wash solution to something more appropriate and also did injections of it? (it's not clear for me, what you did with your wash solutions tested; if only the tubings without the autosampler were cleaned or also the injector parts were included.)

So if there are active metal sites in the autosampler flowpath, it may be hard to exchange that. Depending on the model, you can exchange a steel-needle with one out of PEEK. Maybe even replave some of the tubing with PEEK.
But the Rheodyne and loop may no be possible. (just thinking, because we don't know what system you have).
Passivation with HNO3 maybe an option too, but I guess this will just last a few injections.

And now just one last idea, not knowing if it is even feasible and of help for this method/problem:

What about adding some chelating agent like EDTA (maybe search for something better) to your sample, needle wash solvent or even as mobile phase additive?
The idea behind: if your polyamine is complexing with the metal-ions from the tubing, then something which is stronger may prevent or break the carry-over?
You may want to search for Agilent Poster WP526 from the ASMS 2018, for more inputs (even if the paper is for anions. but you may adapt the idea to your molecules).
Like the inputs from Andy Alpert about the other amines are pointing somehow in the same direction.


Good luck
Hello everybody,

sorry for not responding for quite some time, I've been busy with conducting experiments.

@Andy: I have tried using Trimethylamine as an additive in a seperate method, only for polyamines. It has significantly reduced the amount of carryover in future runs. Unfortunately though, I have not been able to detect any Polyamines using the following eluent composition (any input on that would be nice, if you see a mistake):

A: 80:20 ACN:H2O + 500 mM trimethylamine + 0.5% FA
B: 100 H2O + 500 mM trimethylamine + 0.5% FA

@Hollow:
The system I'm running my experiments on is composed as follows:
LC: Agilent 1290 Infinity II
with: 1290 MCT G7116B column oven; 1290 Multisampler G7167B; 1290 High Speed Pump G7120A
MS/MS: Agilent 6495 LC/TQ

I am indeed speaking of real carryover. Whilst sample injection I'm detecting peaks of up to 1*10^6 counts of area, whilst I detect peaks of up to 2*10^5 counts of area when injecting a blank after the sample. This carryover is pretty much constant over the next up to 5 runs.

I excluded the autosampler by doing multiple no injection runs. I have still had carryover. Furthermore, I've done multiple runs through the bypass instead of the column, where I did not detect any carryover. Also, when using a new column for the first time, carryover was significantly reduced (~3*10^3 counts of area per peak).

I have conducted your proposed experiment with the double cycle. I do indeed see a carryover peak at the expected time if you subtract the first cycle.

As I could exclude the needle as a source for carryover, I have not changed the needle-wash solution. If my assumption, to exclude the needle as a source of carryover is incorrect, I will gladly listen to your input. I have however tried cleaning the whole system with a plethora of washing agents, such as H2O:IPA 50:50, Ammonia pH10, Ammonia pH11, formic acid pH2. None of them worked significantly.

I will talk to our technicians regarding the possibility of replacing some of the tubing with PEEK. Unfortunately, I think that the capillaries right in front of the column aren't replaceable by PEEK-tubing.

I will do some research on your EDTA proposal. Thank you so much for your input!

Best,

Dominik
Have you ruled out the multisampler needle seat? If not, remove the needle seat then connect it directly to the pump and wash at 4-5mL/min for a while, or install a new needle seat. Are you using a needle seat backflush?
Hi Dominik

thank you for the update. Very interesting but not easy problem...

Furthermore, I've done multiple runs through the bypass instead of the column, where I did not detect any carryover.

That in fact points to the column itself, and I guess one can exclude the whole other parts of the system.

Whilst sample injection I'm detecting peaks of up to 1*10^6 counts of area, whilst I detect peaks of up to 2*10^5 counts of area when injecting a blank after the sample. This carryover is pretty much constant over the next up to 5 runs.

That means about half of your sample is sticking on the column!

I have conducted your proposed experiment with the double cycle. I do indeed see a carryover peak at the expected time if you subtract the first cycle.

This is weird for "normal" carry-over, and would only be possible if the compound is sticking quite near the column head and have an inverse retention mechanism, that means stronger retention at higher aqueous mobile phases. Then, when the initial eluent conditions are meet again, another fraction will start its way through the column.

What are the retention times of the spermine/spermidine?
Are they eluting in the isocratic part or close to its end?
If so, did you play with the length of the re-equilibration segment during the multi-cycle-run (no injections between runs)?
And/or what happens if you change the ACN fraction of the inital eluent to 95% and 85%? Do the retention times shift as expected (longer at higher ACN) or is it the other way?

The fact that the area stays the same and does not decrease exponentially, seems more like a saturation effect than a partition effect (e.g. saturated solution inside the pores, that is then eluted and acts like an injection).

All the above ideas points to a really strong interaction with the silica itself and not with some other parts or tubing of the system.
This would also explain, why the same effect is seen on other bare silica columns.

What I've found are the following publications, that may add some more thoughts.

Polyamines like spermidine are also used as templates in the formation of silica nanoparticles and zeoliths.

What if, on a very small scale, the exact same thing happens in the aqueous layer on the silica surface, where probably some dissolved silica gel is present?
What if then some silca nanospheres are formed, which carry some of the polyamine inside and release them only slow or when the spheres are dissolved again under certain mobile phase conditions?

These are only speculations, as I'm not really into silica synthesis. So someone with more expertise in this field may provide further advise.


References:
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