Chromatography Forum

Hi there,
I have a problem with a peak which needs some attention!
I try to separate three compounds which one of them has severe tailing(1.9-2). I use ACN/buffer as mobile phase in a pH range of 3-8. The problem is that none the pHs above give acceptable assymetry factor (As). I didn't try yet intermediate values such as 3.5, 4.5, 5.5 etc, but could it be possible to have a good peak shape at these values of pH? Is there any other factor which i have to consider in order to optimize the assymetry?
(I use phosphate or acetate buffers of 50mM. I can't use more concentrated solutions because i use gradient elution and i noticed a ghost peak appearing when using 100mM of phosphate buffer). Any suggestion is very welcome.
Thanks!
By the way i use a 25mmx4,6mm 5μm column, F=1mL/min and the peak of interest is eluting at 5,2min with 30/70 (v/v) ACN:KH2PO4 50mM.

What kind of stationary phase do you use?
Did you try to run your samples on different stationary phase?

Regards

What kind of stationary phase do you use?
Did you try to run your samples on different stationary phase?

Regards

Hi zokitano,
I use a C18 BDS column. I have a CN column which i am sure it will work better. I have not used it yet. I want to find out what is going on with the C18 stationary phase. Some labs don't have the luxury of using any type of column and stationary phase, so all my efforts aim to solve the problem with the C18 column. Is it possible?

phosphate has a PKa of 2.2 so you better go down. at 3.8 you are above the buffering range, or you have to go up to 7.0 for the second Pka of phosphate.
on the other hand acetate is around 4.8 and 3.8 is under the buffering range you need to go up.
it is best to be within +- 1.5 of the Pka of your buffer for it to be effective as a buffer.

you might need to change to an entire different buffer maybe.
you will need to look at the nature of your compounds, from there you can best decide a good aproach to perfect your method.
you can also "trick" a bit by using a gradient method, that also helps to get a better peak shape

phosphate has a PKa of 2.2 so you better go down. at 3.8 you are above the buffering range, or you have to go up to 7.0 for the second Pka of phosphate.
on the other hand acetate is around 4.8 and 3.8 is under the buffering range you need to go up.
it is best to be within +- 1.5 of the Pka of your buffer for it to be effective as a buffer.

you might need to change to an entire different buffer maybe.
you will need to look at the nature of your compounds, from there you can best decide a good aproach to perfect your method.
you can also "trick" a bit by using a gradient method, that also helps to get a better peak shape

Thanks a lot for the advice...I'll check it out

25mmx4,6mm 5μm column, F=1mL/min and the peak of interest is eluting at 5,2min

Are you sure about the column dimensions? 25 mm is a very short column!

Next question: is the problem peak the first one? If symmetry improves with increasing isocratic retention (i.e., early peaks are worse than later peaks) the first suspect is always extra-column volume, especially with a small column). In your post, you said you were using a gradient, but a bit further you gave elution time under what looked like isocratic conditions.

If your compounds are basic, try taking the pH down further (2 - 2.5). Even with a base-deactivated silica, you can see some ionization of silanols at intermediate pH. As unmgvar pointed out, that will also put you into the working pH range for phosphate buffer.

You didn't say how much you were injecting. Is there a chance you are simply overloading that peak (quite possible if you don't have a good chromophore). Try injecting 1/10 as much. If the peak shape improves and retention time increases, then you were overloading.

25mmx4,6mm 5μm column, F=1mL/min and the peak of interest is eluting at 5,2min

Are you sure about the column dimensions? 25 mm is a very short column!

Next question: is the problem peak the first one? If symmetry improves with increasing isocratic retention (i.e., early peaks are worse than later peaks) the first suspect is always extra-column volume, especially with a small column). In your post, you said you were using a gradient, but a bit further you gave elution time under what looked like isocratic conditions.

If your compounds are basic, try taking the pH down further (2 - 2.5). Even with a base-deactivated silica, you can see some ionization of silanols at intermediate pH. As unmgvar pointed out, that will also put you into the working pH range for phosphate buffer.

You didn't say how much you were injecting. Is there a chance you are simply overloading that peak (quite possible if you don't have a good chromophore). Try injecting 1/10 as much. If the peak shape improves and retention time increases, then you were overloading.

I'm really sorry, for the mistake...i was too tired to notice!I use a 250mmx4,6mm column. The conditions are as such:the total elution is a gradient elution. But until the first peak appears, the conditions are isocratic. So i thought that i don't care about the gradient, but what it happens in the first 5min.I have no clue about the pKa of the compound. The problem is that when working at low pH the compound elutes very quickly, and i can't see the peak...I decided to scan the pH range and i found that as pH increases the retention time of the peak increases too. With phosphate buffer i worked in a pH of 3, 7 and 8. Doesn't have enough buffer capacity in that values of pH?As for the injection, i inject 20μL.

Still not yet clear what the problem could be.

1. It is not clear how much analyte you are injecting, and if you are overloading the column. Either give the amount of analyte injected in mg, or at least tell us what the UV response is (20μL is insufficient info).

2. Based on your observation of the retention change, you are getting a higher ionization with decreasing pH, which indicates a weak base in your molecule (an aniline function or a pyridine function). This is per se not a problem, but you might be better off with a more modern packing based on a high-purity silica or a hybrid packing.

3. You did not say anything about the sample solvent. Using an incorrect sample solvent can use all kinds of peak distortions, especially before the onset of a gradient.

Try adding triethylamine or another base to the mobile phase(start with 1ml/litre) this should prevent tailing of a basic compound.