Right-triangular peaks - What's the cause?

[peptidemetdev]

I am doing a separation of TFA and Acetate on a reversed phase column, and I notice that as I increase the amount injected of either compound the peak becomes increasingly right-triangular. At lower concentrations/injection volumes, the peaks are fairly gaussian, but as I increase, the front end becomes more and more vertical, and the back end becomes more and more sloped (a linear slope, not really a typical tail). Also, retention seems to decrease as the peak shape becomes more right-triangular. However, despite the changes, linearity is still very good.

Does anyone know what causes such a change in peak shape? Can anyone recommend a good resource that might give some explainations for various changes in peak shape and how they could be interpreted?

Thanks.

[mbicking]

Triangular and "shark fin" shapes almost always indicate overload, and are often accompanied by a reduction in retention. You have already done the experiment to prove this - when you decrease concentration, the peak shape improves.

You should find a good troubleshooting resource. Dolan and Snyder have an excellent book, but there are several others as well. Or, you may want to attend a one or two day course on HPLC that covers troubleshooting. Again, there are several options available.

[DR]

One other (though frequently less obvious) symptom of overload is found at the tops fo peaks - if you have several points across the top that are at the same height from the baseline, you've saturated the detector (or the A/D's input stage).

[peptidemetdev]

Thank you for your replies.

Would decreasing the particle size of the packing reduce or increase the over-loading effects?

I'm not positive what the retention characteristics actually are for this separation, but I do know that the plate count for the TFA is much lower than the plate count for the Acetate, and that it seems to suffer from the over-loading effect at a much lower concentration than the Acetate. I'm thinking about buying a smaller column to further increase the sensitivity of the method, but I'm not sure if moving to a 3µm packing from a 5µm one is a good idea or not.

Any thoughts?

[tom jupille]

Two kinds of overload:
- detector (flat top peaks)
- chromatographic (tailing peaks + decrease in retention time).

You are seeing chromatographic overload. There is no "absolute" level at which overload begins; it depends on the details of the chemical interaction between your analyte(s) and stationary phase. In a case where the interaction is with specific active sites, overload can occur at suprisingly low mass levels (and yes, it can be different for different mobile phase compositions).

Other things being equal, the particle size has little if any effect on loading capacity, since most of the stationary phase surface is inside the pores of the packing. A higher surface-area (smaller pore diameter) packing will increase the loading capacity somewhat, but from your description of the peak shape, I doubt that it would be nearly enough.

Your best bet would be a more sensitive detector (or GC, for that matter).

[danko]

Hi Peptidemetdev,

Maybe you should try the ion-exchange mode (anion- exchange to be more precise).
It’s a straight forward and quite rewording technique.

Best Regards

[Uwe Neue]

The overload on a column is a function of the chromatographic conditions. A change in the column dimensions won't help.

Please tell us more about your analytes, and the concentration and pH of the buffers (?) that you have used. There are ways to improve the lodability drastically, if your analytes are ionic, but it may require to change the chromatographic conditions.

[peptidemetdev]

The overload on a column is a function of the chromatographic conditions. A change in the column dimensions won't help.

Please tell us more about your analytes, and the concentration and pH of the buffers (?) that you have used. There are ways to improve the lodability drastically, if your analytes are ionic, but it may require to change the chromatographic conditions.

The method is a single, quick method used to determine TFA/Acetate content of lyopholized peptide. I've determined that the LOD is around 150-200ng for TFA which is plenty for quantifying the TFA content of a TFA salt, but not sensitive enough for determining residual TFA content of an acetate salt (which we've seen as low as a hundred ppm), which I figured worked out to be a detection limit of around 5-15ng depending on how much sample I was going to use in my preps. I did a few tests and determined that the detector noise was independent of flow rate, so I figured I could increase the signal to noise ratio by decreasing the flow rate substantially. I could also increase the signal by increasing the amount injected, but that's where I began to run into the loss of efficiency.

I do not have a GC available for use, and my goal was to modify an existing internal test to use when sending to an outside agency was prohibitively expensive for the project. We used to use a Hamilton ion exclusion column for acetate content, but we adopted this method in order to do TFA content as well.

The conditions are as follows:

C18 column (4.6x250mm, 5µm packing). Currently using a YMC ODS-A 120 angstrom
Isocratic condition, 2% MeOH in 0.1M sodium phosphate pH ~2
Flow is 1.5mL/min
Detection is UV at 210nm

The high end of the calibration curve for the original method is around 200µg, and the k' for TFA is around 1, with the k' for Acetate around 2. TP for TFA is about 4000, while TP for Acetate is around 15000. Resolution is generally around 4, all measured by the HPLC software.

I'd considered ordering a smaller version of the same column with 3µm beads to try and increase sensitivity a bit more, but if pore size would make a bigger difference, maybe I will try an Agilent column I have on hand that has an 80 angstrom pore size.

[mbicking]

You could also try a "polar embedded phase" such as the Agilent Bonus RP, the Supelco RP Amide, the Mac-Mod Prontosil EPS, or the equivalent from Waters (name escapes me right, but Uwe can supply it).

These columns tend to retain acids somewhat, which might help you, and they work well in highly aqueous mobile phases. I would use a mobile phase of 0.1 % phosphoric acid (no real need for a buffer) and 0 - 2% methanol .

Another problem you have with overloading is the fact that neither analyte absorbs much at 210, especially TFA. You could try 205 and get a little bit more sensitivity, but the baseline might become less stable.

[peptidemetdev]

I took your advice, mbicking, and was surprised to see the response of the TFA shoot way up!

210 seems to be an absorbance max for acetic acid, but it doesn't decrease very fast as I lower the wavelength.

The last injection I tried was at 190nm. The baseline is starting to go a little bit crazy, to the point where it looks like the Acetate peak is on the TFA peak tail (although the software still shows a resolution of 4). The response of the TFA has increased by a factor of 13 (although the response of the Acetate is only 2/3 what it used to be), and I bet I still haven't come close to hitting the absorbance max. The problem is that the background absorbance is getting too high, and I'll hit the detector limit soon, so I think I'm going to stop at 190.

[mbicking]

I would recommend that you stay above 190 nm. This is below the "UV cutoff" for your solvents, meaning the absorbance due to the solvents is 1 (or greater). With methanol in the mobile phase, there won't be much light reaching the detector, and your results will probably not be as precise.

If you could switch to acetonitrile rather than methanol, your absorbance would be less, but I would still try to operate at 200 nm. If you need more sensitivity, then investigate a column with more theoretical plates.

[peptidemetdev]

Is there anything terribly wrong with using 190nm as long as the peak heights never reach detection limits (which would be around 0.9AU, taking into account the background absorbtion of around 1.1 AU), and results are consistent enough to achieve a good linear concentration curve?

Is the Methanol the only thing that absorbs so strongly? I do have some 100% aqueous compatable columns on hand, maybe I'll investigate using them instead.

[mbicking]

The methanol is the primary source of absorbance in your system, but everything is absorbing at 190, including water. One problem is that you have created a nearly universal detector. Since just about everything is going to absorb at 190, you may see extra peaks. If these interfere, then you have new problems. I suspect things would be better at 195 or 200 nm. But if you have good chromatography and linearity, you could indeed use these conditions. But investigate precision carefully.

If you really need sensitivity, I would go back to my suggestion of finding a better column. And, if you switch to a smaller bore column (3 mm is my preference), you can slow the flow to 0.6 mL/min. If you are lucky, keep the same injection volume and you will see an increase in sensitivity. You may have to decrease injection volume somewhat if the peak shape is poor.

[Uwe Neue]

I am glad to see that you are making progress. Did you take Merlin's advice and used some phosphoric acid in the mobile phase?

[peptidemetdev]

merlin: I was not seeing any interfering peaks, and I figured that a 100µL injection at 4.6mm did lower the plate count, but I bet, like you say, with a more appropriate column, I could get away with an increase in volume without distorting the results too bad. I have a few smaller bore columns on hand (2.0x100 3.5µm Phenomenex Gemini, 2.0x100 5µm Phenomenex Jupiter, and 3.0x100 3.5µm Waters Sunfire) I could try without spending any additional money, so maybe I'll begin there. I also have a 3.0x250mm YMCBasic column, but I have a feeling retention/resolution won't be as good on that column for this method.

Uwe: I developed a pretty simple method using 0.2% phosphoric acid in water, a few months back, to analyze Succinic Acid on an aqueous column (the Gemini). I think I might play with that and see if I can get good separation with TFA and Acetate, as it seems to me like they should be easier to retain than succinic acid was. I do agree with him that it doesn't seem like there's any reason to buffer the mobile phase, as long as the pH remains below 2.5 or so (pKa of Acetic Acid is 4.5, IIRC, and that's probably what allows the separation on a RP column).

Thank you all, again, for your help!