The perfect peak shape: Five solutions to peak tailing problems

For chromatographers, there is no sight more beautiful to behold than perfectly symmetrical peak shapes! But how do we even measure peak shape symmetry. And do perfect peaks really exist? Certainly not when you have peak tailing problems! Read on to find out more about perfectly symmetrical chromatography peaks and five ways to achieve them by avoiding peak tailing.

My wife and I were out for a stroll the other day when she noticed a young woman wearing a very interesting skirt made of different fabrics that were asymmetrically cut. My wife commented that she liked the skirt but noted that her mother would have absolutely hated it. Apparently, it was very important to have perfectly cut, straight, symmetrical skirts back in the days. She argued that symmetry is just more appealing to humans.

Well, beauty is in the eye of the beholder. I don’t care much for symmetrical skirts, symmetrical faces or symmetrical flowers. But as a scientist, I do like my symmetry. Come on, who didn’t get excited this week about the possible detection of an axion, the lightest predicted supersymmetry particle, underneath an Italian mountain last month?

And as a chromatographer, oh boy. There is nothing like those perfectly symmetrical peaks in a chromatogram to get me going!

We all know perfect symmetrical peak shapes obtain the best results, but sadly, reality hardly ever results in perfect situations. How about we go on to explore peak shapes in a bit more detail?

I suggest we start off by reviewing how the symmetry of a peak is measured. There are two main ways to do this.

The first method is based on the tailing factor ( Tf ). Here, you measure the peak width at 5% of the peak height above the baseline. This approach is often used in the pharmaceutical world.

chromatography, peak shape, tailing factor

The other method relies on the asymmetry factor ( As ). Here you measure at 10% of peak height above the baseline.

chromatography, peak shapes, asymmetry factor

A peak with:

Tf or As  = 1 is perfectly symmetrical.

So now we know how to measure peak symmetry. But as I already mentioned, like people, skirts and flowers, chromatography peaks come in all shapes and sizes. Unfortunately, in some cases, this can be rather problematic. I’d like to go on to discuss one frequently encountered phenomenon with you here, mainly peak tailing!

Back to our chromatogram, peaks with:

Tf or As  >1 are tailing.

What causes peak tailing?

Well, there are various possible causes of this peak shape.

chromatography, peak tailing, chromatogram, peak shape

Cause 1: Firstly, tailing can occur when secondary interactions take place. As a result, not all molecules travel through the column at the same speed and this causing tailing at the peak.

Possible Solutions: To remedy this, you could try to lower the pH of the liquids so that silanol ionization is suppressed (pH 3). Reducing the pH keeps the silanols in protonated form, where interaction with polar compounds is minimized. This, in turn, has a positive effect on your peak shape.

Alternatively, you could use packing material with better end-capping to reduce the surface activity. Lastly, you could try adding buffers to the mobile phase running through your chromatography system. For purification purposes, these buffers should be volatile, so that they can easily evaporate with the mobile phase after sample collection.

chromatography, silanols, protonated form, peak shape silanols

Cause 2: Tailing can occur due to packing bed deformation. The creation of a void at the inlet of the column or the presence of channels in the packing bed can initially lead to peak tailing. Eventually, this phenomenon could lead to peak splitting, another problematic shape I’ll discuss in a future post.

Additionally, collection of particles at the inlet frit can lead to peak tailing.

Possible solutions: Apply extreme care when self-packing columns or purchase pre-packed consumables if possible. Filtration of the samples prior to introduction on the column can help prevent peak tailing resulting from collection of particles at the inlet frit.

Cause 3: Tailing can occur due to column overload

Possible solutions: You could try using a bigger column or decreasing the sample amounts introduced to the column to eliminate the problem.

Cause 4: Sometimes, the presence of excessive extra column dead volume can result in peak tailing. This problem usually effects the peak shape of the early eluting peaks. Later eluting compounds will be more influenced by the packing material itself as these reside longer in the material.

Possible solutions: Again, exercise extra caution when pre-packing your column.

Cause 5: The purity of the packing material can also influence peak shape. Contaminants, such as trace metals, in the material can enhance secondary interactions, which will lead to peak tailing.

Possible solutions: I would recommend using the purest packing material possible to help fix this problem.

So there you have it. Five causes of peak tailing and five remedies to help you leave this problematic peak shape behind. Unfortunately, I also have some bad news. There are more nasty peak shapes that can occur during your purifications. But I would like to leave you with some good news. I will offer you some ideas on how to eliminate other asymmetrical peaks in a future post. And I promise, I will not keep you waiting long.

Till next time,

The Signature of Bart Denoulet at Bart's Blog