Right-triangular peaks - What's the cause?

[Uwe Neue]

I do not know why this was posted here, since there is no connection to the question. However, smaller diameter columns do not offer a benefit in efficiency.

To peptidemetdev: If the surface chemistry of a packing is not known, it is difficult to sort out why it works, or why it does not work...

[HW Mueller]

And it is especilly difficult to sort out why it works if one uses nearly 100% aqu. phase on what appeared to be a HILIC column, then goes to nearly 100% organic (or am I confused once more?).
Also, did you use strongly acidic conditions (H2SO4?) to try to get anion exclusion on an IX?

[danko]

Hi peptidemetdev,

Purchasing a new column (especially one as expensive as a mixed mode column) is, unfortunately, a tough sell right now.

Understandable argument.

………and it really seems like they have really poor capacity and peak shape (the peaks are symmetrical, but extraordinarily wide).

In the Ion Exchange technique, the loading capacity is quite dependable on the buffer pH. That is especially true for strong exchangers. The fact that the peaks are symmetrical sounds promising. So, you “onlyâ€

[SIELC_Tech]

Before starting a project, it always makes sense to evaluate tools available to complete it, whether you are digging a hole, building a deck, or developing an analytical method. With wrong tools you will spend a very long time and outcome of your efforts might be mediocre at best. Some times there is a need to get a new tool. Very often chemists in the lab would say that they don't have particular column and they rather use what they have in the lab. Nobody ever calculates the "human" impact on the cost of analysis. If chemist spends two weeks developing a method without success the cost to the company is much more than buying a new column (with average cost of chemist to the company being even $50/h results in $4,000 cost for two weeks development, the average cost of employing chemist in the US Pharma company is around $120/h).

Anyways, to make log story short. As I said, mixed-mode approach is one of the best approaches in analysis of ionic hydrophilic and hydrophobic molecules. I just spend an hour setting up sequence to screen UV method for TFA and here are the results on mixed-mode Obelisc N column, Atlantis T3 reverse phase column and Sequant Hilic.

HPLC Analysis of TFA
http://www.sielc.com/application_230.html

There is not too much retention on Atlantis T3 and peak shape is the same as on your other RP columns, peak shape and plate count are much worse than on Obelisc N mixed-mode column. We have not tried IP approach as this can be a better method in terms of retention time. With reverse phase and HILIC you don't have too much room to play with ACN and buffer. At high ACN in RP you loose all retention of TFA and at low ACN and buffer concentration you are suffering from poor peak shape. In HILIC you probably can use various UV transparent buffers at higher pH and try to see if this works. We did not try to optimize HILIC mode and HILIC experts are welcome to provide link to the method.

Regards,

Vlad

P.S. You can inject 5, 10 uL at 3 mg/ml without overloading Obelisc N column. We did not try to inject 10-50/mg/ml

[Uwe Neue]

Vlad:

Why did you use 5% acetonitrile on the Atlantis T3??? No need, you can run this column in 100% water, and you will get MUCH more retention.

Also, if you just use a decent concentration of phosphoric acid, such as 50 mM, you will get a decent peak shape for the TFA. Your very dilute sulfuric acid is overloaded, which is why you get the tailing peak

[peptidemetdev]

SIELC_Tech: I've found that my method gives a plate count of around 12000 for TFA at about 3.6µg injected (0.1848 mg/mL, 20µL injected, Agilent Zorbax Eclipse C18, 5µm/80A). I'm not sure at which concentration the peak shape begins to be effected, but the peak is right triangular with a plate count of around 4500 at around 18.48µg injected. Also, the sort of tailing you see on the Atlantis isn't quite the same as what I've been trying to describe. The tailing I see is much more of a right-triangular peak, with the asymmetry pretty much completely distributed from top to bottom of the peak, not just localized at the bottom.

Uwe: I wouldn't be so sure about a decrease in ACN increasing the retention. The MeOH in the method I describe appears to do nothing more than improve the peak shape for the TFA. Retention times are not affected much by eliminating the MeOH, and in fact, the retention time for the TFA appears to shift to the left slightly when I move to a 100% aqueous mobile phase.

Anyhow, thank you all, again, for your help and suggestions.

[SIELC_Tech]

Quick test showed that neither increase in amount of phosphoric acid or decrease of ACN improved retention time or peak shape on Atlantis T3 column (pictures will be posted later). We are injecting significant amount on Obelisc N column, when 5 uL of 3 mg/ml was injected in RP we got exactly the same peak you have on Zorbax (right-triangular peak))

[Keaka]

I've been running an almost identical method for TFA/Acetate separation (your original method) on a standard C18 column and found the performance okay for my own purposes (injection volume around 50 µL).

I haven't tried anything higher, but I'm curious if in the analysis of TFA/acetate content in your peptides that your calculated % of counter-ion is biased low as that is what I've been consistently seeing.

For lower detection limits I'm afraid ion exchange is the only way to go.

[peptidemetdev]

I haven't tried anything higher, but I'm curious if in the analysis of TFA/acetate content in your peptides that your calculated % of counter-ion is biased low as that is what I've been consistently seeing.

For lower detection limits I'm afraid ion exchange is the only way to go.

I can't say for certain, but there does not appear to be any significant bias one way or the other for our results. Our reagents aren't particularly well-controlled, though. For example, we don't attempt to recover a secondary standard to verify that our concentration curve is accurate, and our Sodium Acetate has been kept in a poorly controlled, room temperature dessicator for about 4 or so years. Our results, so far, have been comparable to what we saw with the ion exclusion column, which were also similar to an outside testing facility we had used in the past.

Which column do you use, if you do not mind sharing?

SIELC_Tech: It sounds to me as though the Obelisc N column does not perform significantly better than a properly selected C18 column (and, in fact, may perform worse based upon those plate counts you linked to). The increase in retention probably looks good to an auditor, though. I know they don't like to see retention factors anywhere near 1.

In either case, have you attempted a separation of acetate and TFA? Based upon the patterns I've noticed, I would suspect that, with the methods you displayed, you would either not see any retention, or the resolution would be poor. I would be interested in seeing if this is actually the case. That first example you listed actually looks very similar in terms of retention to the results I saw on the Epic HILIC column with a pH 6 ammonium phosphate mobile phase (no organic modifier).

[SIELC_Tech]

Plates are per column (8000 for 150 mm column).
We have not tried AcOH and TFA, but might come back to it later. Obelisc N provides perfect peak shape and good plate count and allows you to analyze wide range of concentrations. We tried 3 different RP columns with the same results (poor peak shape and low plate count).
It would be nice if you can post your pictures here for comparison.

[HW Mueller]

Vlad, it seems that the only interesting experiment in this context would be to do AcOH and TFA together.


Peptid....., it is totally contrary to my experience not to see any effect when changing the organic modifier or the ionic strength under similar situations. Could it be that your overloading problem is masking things? Anyway I am confused about what are results of overloading and what of real chromatography here.

[peptidemetdev]

Vlad: My appologies, you are right, I did not consider the effect of length. Such high peak counts with a shorter column is pretty impressive. Your results with the TFA are superior to what I've seen. At about 20µg, the pH 6 ammonium phosphate on the hilic column had a plate count of about 11000, and an asymmetry of 1.7.

At about 3.6µg, the Zorbax using the original mobile 2%MeOH/buffer mobile phase had a plate count of 10000, and an assymetry of 1.7.

Both columns have a length of 25cm.

At any rate, HW Mueller is right about a separation of TFA/HOAc, except that I'd say another experiment of interest would be a LOQ test with the TFA. With the lowest end of my calibration curve being around 10ng, I can achieve a good degree of linearity (3 levels, 2 injections per level, R² > 0.999).

Another curiousity I'd have is whether or not peptides would be retained under the conditions you listed. This is simply a matter of convenience. The low-organic conditions used in the original method allow multiple injections to be done in a short period of time, followed by a long wash/regeneration cycle at the end of a sequence. The accumulation of small amounts of peptide at the head of the column does not negatively impact performance (for whatever reason, our prior methodology using the ion exchange column never called for a column wash, but I guess that explains why they saw the columns go south after only a few hundred injections a piece).