injection 1: peak ok/injection 10: tailing and smaller area

[LCFlo]

Dear all,

I am using a method for separating a main compound and three impurities using an ACE 3 C18 column (150 x 4.6). It is an ion-pairing method. When I use the column at first time, it works well. After 10 injections (sometimes after 70), the peak shape of the last impurity becomes awful. Due to the fact that I don't have to quantify it, the phenomenon is not a "killer". Nevertheless I thought about the reasons:

- column reproducibility
- ion-pair reagent reproducibility and power

When the impurity beak becomes awful (tailing factor of 10 or more), the peak of the main compound keeps unchanged, also its area remains constant. But the peak area of the impurity decreases from about 80'000 Units (1st injection, good peak shape) down to about 15'000 (bad peak shape).

What might be the reason for the loss of area with a change of the peak shape? I thought that the peak area should not change with its shape change.

Thank you for every hint

Florian

[Uwe Neue]

Mobile phase? Conditions?

[LCFlo]

26% of a solution of sodium octane sulfonate, 2 g/L, pH 3.5 (CH3COOH)
74% of MeOH

Flow = 1.0 mL/min
Temp. = 30°C
Wavelength = 240 nm
Path length of detector cell: 2.5 cm
Injection volume: 5 uL, sample dissolved in mobile phase[/list]

[Uwe Neue]

I don't understand (yet) why the peak turns bad. However, a terribly tailing peak is more difficult to integrate, and you could loose all the missing peak area in the peak tail.

Are you buying ion-pair reagent for HPLC, or reagent grade octane sulfonate?

[LCFlo]

I tried to integrate the peak in a way that everybody would say that the resulting area is too high. Nevertheless, I got only a percentage of the initial area.

Regarding ion-pairing reagents: I buy it from a supplier which delivered me many years with reagents from Scharlau (HPLC grade). But due to increasing prices, he changed to Reuss-Chemie. I have to clarify what quality this really is.

[danko]

It might just be that the degradation peak undergoes further degradation (i.e. becoming 2 or more additional compounds/peaks). So, some of the stuff could cause the tailing (i.e. being 2 or more not separated peaks) while another fraction could just elute unretained (with the void volume).

One possible examination of that hypothesis could be: Running several injections of the same solution until the phenomenon is observed and then injecting a freshly prepared standard/SST solution that either would confirm or discard the possibly of chromatography deterioration.

Best Regards

[LCFlo]

That is what I tried first: I checked with a freshly prepared solution, whether the phenomenon rises again. And it did

[danko]

OK. What do you do then to “resetâ€

[LCFlo]

Usually, I do nothing because I do not have to quantify the peak, I only have to wait for its elution. But the chromatograms obtained cannot be shown to authorities because they have a problem with the peak shape independent on the fact that I do not care about the peak.

When also the mein peak begins to tail (after 200 - 250 injections), I change the column. Then, it might be that the first or second injection is bad regarding the peak shape. Other times, I can perform about 70 injections with good peak shape.

Often, I thought that the equilibration time might be too short (ion-pairing method!). Thus, I waited 5 to 6 hours or longer with the first injection. But I got some bad peaks also after this equilibration time. Additionally, the peak shape should become better within a sequence starting with bad peak shape. This was never the case.

Some other phenomenon: I reserved two lots of packaging material, which I tested. They gave a good peak shape. Also after 70 injections what is ok for me. Using such "same-lot-columns" now, I have the described problem after 2 or 3 injections. Thus, I assume it might be:

- the packing quality (and its reproducibility, respectively) or
- the performance of the ion-pairing reagent (and its reproducibility, respectively)*

* the low price of it might additionally justify this assumption

Do you think in similar way?

Regards

Florian

[danko]

I don’t think the low price (indicating, but not necessarily low quality) of the ion pairing agent is the problem here. If it was impure or something like that, you would see consistent impurity peaks stemming from the chemical itself.

As you present the story, the problem appears and disappears (the first injections are not satisfactory) randomly. So, there must be some kind of instability (maybe pH?)

You forgot to share one important information: When you employ a new (freshly prepared) solution, is everything OK then (i.e. normal/desirable chromatography) even though the “not freshly prepared solutionâ€

[LCFlo]

Problems can arise using a new colum independent on the solution I inject (old/fresh); if the first injection is bad, the peak shape does not became better with longer equilibration times.

I ordered ion-pairing reagents from other suppliers to compare the chromatograms with the same column and injected solution. Hope they will be delivered soon.
I already tried an alternative lot of the current ion-pairing reagent supplier. The problem remained the same.

Regarding pH: there is a peak pair in the chromatogram which resolution is strongly dependent on pH. But I never had a problem with this resolution (none of these peaks is my "problem peak")

[Hollow]

Hi Florian

Maybe it's really related to the ion pair reagent.

We observed also some "strange" behaviour when we changed the reagent grade. With the cheaper, not specially labeled as "ion-pair grade" reagent, our peak eluted earlier with every injcetion.
Switching back to the original ion pair reagent made the phenomenon gone.
See "my" thread viewtopic.php?t=10064&highlight=catecholamines
We bougth both reagents from Fluka.

Hope you're able to solve your problem soon.

[LCFlo]

Hi Hollow,

I just read the thread you linked: sounds very interesting and let me hope that the reason of my problems might be the quality of the ion-pairing reagent. If yes, I will have to talk to our financial department in order to force them to provide any additionall Swiss Francs for lab next year

Regards

Florian

[mohan_2008]

LCFlo,

Ion pairing chemistry is a complex phenomenon involving several equilibration processes.

It appears that your last impurity is not retained through Ion-pairing interaction however the main compound is. It is reflected by a consistent peak area of the main compound but not of the impurity (after several injections).

After 70 injections, if your impurity is retained by ion pair interactions, you should obtain a consistent peak shape - but it is not happening which confirms that the impurity is not held by ion-pair interaction.

I don't think it is going to help trying to trouble shoot the impurity peak, as your method may not allow this to happen.

When developing your ion-pair method - first evaluate any peaks through the regular RP interactions followed by ion pair method (only if necessary). Since neutral species may act erratically under ion-pair conditions (mixed mode interactions - Part Ionpair/Part Reversed-phase).

[HW Mueller]

The problem here is that we don´t have any idea which species are specially susceptible to ion pairing. How a neutral species should "partly" be influened by ion pair interaction is a riddle to me.