Chromatography Forum

In the QC lab. where I am working was transfered a method for related substances of an drug product using HPLC.
The method parameters are: Agilent Eclipse XDB C18 (250 mm x 4.6 mm), UV detection and the mobile phase is 95:5 buffer pH 7.2: ACN.
Buffer preparation: 0.69 g sodium dihydrogen phosphate, 0.64 g of tetrabutylammonium bromide and 0.55 g of EDTA disodium salt in 1 l of water. Ph adjusted to 7.2
We were runing this sistem few times and system suitability was OK (tailing less than 1.5). For few weeks we obtained poor peak shape (tailing more than 1.5- usualy 1.8 ). We used the old column and a new column too. The results was the same: tailing 1.8 or more. The diluent used is water.
Please advice how can I get a good tailing (less than 1.5)

First thing to look at: if there is more than one peak on the chromatogram, do they all show the same problem (if you only have one peak, try injecting a mixed standard that *does* have multiple peaks.

If all the peaks show the same kind of tailing, there is a high probability that the problem is a distorted flow profile at the column inlet. A partially plugged frit (unlikely on two columns), a bad guard cartridge (if you were using one) or even a tube end that doesn't go all the way down into the fitting (especially likely with PEEK fittings).

On the other hand, if some peaks tail while others look OK, it's probably a chemistry problem. If only the big peak tails, it's probably overload.

Good afternoon.

Indeed, the high value of the tail factor may be due to the chemical nature of matter,[is his fate]. Recommend to try to dissolve it in the mobile phase and check how to change the tail factor (the degree of protonation of the substance for the mobile phase and water can vary significantly and this can lead to an increase in retention).

I may casually read your message. Tail factor 1.8 for the impurity? Or for the basic substance?
If a basic substance - then "pofig" [slang- is not affected], its peak is huge and can be calculated with reasonable accuracy regardless of the length of the tail.

Thank you for your support and ideas.

I have injected on this sistem a standard solution that contains only one compound: the active substance of the drug. So I have only one peak.
I do not used a guard cartrige and I have used a new column and the problem it is still the same. This make me think it is not a plugged frit. I have injected a sample solution (in this solution the main peak is bigger than in standard solution but the peak is with great tail and had a shoulder)- this make me think it is a chemistry problem. From my experience in HPLC this kind of problems apears by using buffers on basic pH and by using a mobile phase in great % aquous.
I can not ignore this tail because "the system is suitable for anaysis, if and only if the tailing factor for A peak is not more than 1.5".
I have injected standard prepared in mobile phase and the problem is the same.
Thank you again.
regards, Mangu

Sometimes, another small component (for whatever reason) co-eluting with the main analyte with a slightly longer retention time (if injected alone) could make the peak of interest appear to be like a tailing peak. This interference (if exists) maybe due to an impure std, or come from mobile phase.

Sounds like a "chemical problem" allright. What type of analyte do you have? Could be that your pH varies and/or the ionic strength is too low. What is the sample/standard solvent? Has it always been the same?

I have an analyte that has five degrees of ionisations. The five pKa values for analyte are :
pK1 = 1.6 ± 0.2, pK2 = 2.2 ± 0.2, pK3 = 5.9 ± 0.1, pK4 = 7.1 ± 0.1 and pK5 = 11.7 ± 0.3.
The solvent used for sample/standard is water.
It has not always been the same. Few months ago we have made this analysis with no problem and we got 1.1 tailing. It is strange that on new columns the problem is the same.

Having the mobile phase pH close to a pKa of an analyte is always risky in terms of retention and peak shape reproducibility. Try tweaking the pH 0.2 units one way or the other and see if that makes a difference.

If the problem occurred suddenly, did it coincide with a particular event (e.g., new batch of buffer salt? new pH electrode? change in lab temperature? different instrument?)? If so, that may provide a clue.

I was asking whether you made a slight change in method, the same question which Tom asked now.
It could well be that you have a pH incompatibility with the samples in water.