Chromatography Forum

Dear members of this forum,

I have recently a problem with a determination of loperamide in a liquid formulation. The problem is an important fronting but not tailing and I do not believe that the reason will be the column. The water mobile phase is a mixture of hidrocloric acid and trietylamine in order to simulate trietylamine hidrochloride and 0.5 ml of phosphoric acid and using the trietylamine to reach to 3.00 (total volume is 500 ml). The total mobile is phase is 55 % of the phase detailed and 45 % of acetonitrile. When I saw the mobile phase I do not like the hidrochloric acid, the important content of trietilamine added (about 10 ml in 500 ml) and the quantity of phosphoric acid is low (not reach to 10 mM).
I think the the fronting would be the important quantity of trietyalmine or the low buffer capacity. I need your opinion in order to reach a solution because I think the column is not the reason.
Thanks in advance for your help,

Diego Delmonte

how much do you inject? Have you tried injecting less?

And what is the solvent composition of your sample?

I dissolve the sample in mobile phase and I try to inject less sample because in all troubleshooting about HPLC the main reason of the fronting is big sample injected.
Thanks and I will be waiting other opinions,

Diego Delmonte

I have seen fronting from various causes. One is simply injecting too large of a sample volume. Another is injecting too concentrated a sample. Another is a mobile phase that has inadequate buffering capacity. In all three cases, injecting a smaller or less concentrated sample improves the chromatography. Of course, you may have good reason for injecting that much. You can try increasing the concentration of buffer in the mobile phase, or using a different buffer. If that does not work, you may need to use a different type of column that has better loading capacity; to a first approximation, loadability increases with surface area of the silica and the cross-section area of the column.

I agree with you about the hydrochloric acid. It is harsh on your HPLC system at pH 3, and you can safely substitute other acids.

I want to know it the reason of the fronting could be a problem with the column because in every articles about the regeneration is mentioned the reasons of regeneration is an important tailing and high back pressure.
If the fronting is an evidence of a problem of the column as preference ways into the column, I want to know ways to regenerate.
Thanks for all and I want to know more opinions,

Diego Delmonte

There are three forms of column failure that can cause fronting.
- A void at the top of the column is the most common. Sometimes you can refill the void with stationary phase; you have nothing to lose by trying.
- Channelling is not common because it is a manufacturing defect; there is no way to fix this.
- Partial dewetting can also cause fronting. This can be treated. Wash the column with degassed acetone, then methanol, then your mobile phase.

To add to Mark's comment: if it is a column failure problem, you will see fronting on all peaks, no matter what you inject. Check this before you try to check for a column failure problem!

Dear Diego,
I think that your problem could it be in analyte and application stationary/mobile phase. I suppose you are using C18 phase. Try to use trifluoacetic acid or heptafluorobutyric acid (10 – 20 mM) instead of hydrochloric (phosphoric acid) with TEA (adjust to pH 2.5-3.0). As mentioned above the problem can be in too high volume injection or high concentration. Did you try really injection of very low concentration of loperamide?

Dear members of this forum,

Thanks for your help. I try to do all your advices but I suppose the problem is the column and not the analyte because I inject a standard of sertraline which is analysed with the same column and the peak has the same fronting. I think the buffer capacity, the medium of dissolution the drug and the concentration is not the problem. I start to work in the direction of the presence of a void and a dewett problem. First of all I will do a cycle of washing the column.
Thanks again and I will be waiting more advices,

Diego Delmonte