Chromatography Forum

Dear All,

I am currently developing a method to analyse acetamiprid residue in vegetable oil sample using LCMSMS.

I have a problem with my analyte peak which is jagged and tailing.
Below are the setting of my instrument.

Solvent 70% MeOH with 5mM HCOOH, 30% H2O
Flow Rate 1ml/min
Column: C18, 50 x 2 mm, 5um.

This problem only happend during the injection of real sample and not during the standard injection.

I also found out that if the % of MeOH increase, tailing and jagged effect decrease.

Please help me. Thanks in advance.

Hi Kenny

This is just a guess because you do not tell us what your samples and standards are dissolved in, but peak distortions can be caused by injections in stronger solvents thnat the mobile phase (in this case probably 100% methanol ?).

Peter

Just for fun, take a look at a mass from the background, such as a solvent impurity, column bleed, etc. It should be constant. Do you see jagged modulation in the background when you inject a real sample? If so, you have ion suppression in the electrospray interface caused by a co-eluting component.

Thanks Peter, you are right, I am using 100% methanol as the solvent to dissolve my analyte.

I found out, at 75% methanol, the tailing and jagged effects were reduced, but I don't want to use that strong solvent, any suggestions?

By the way, can you explain to me why was the methanol that gave the problem of jagged and tailing peak?

Hi Kenny

The solution (pun !!) is to dissolve your samples and standards in a water methanol mixture with the same (or lower) % methanol as the start of your mobile phase gradient.

The peak distortions are caused by the methanol injection mixing unevenly with the mobile phase at the head of the column. Analyte that is in a region of high methanol concentration moves ahead of the rest of the peak, causing distortion on the front edge of the peak.

Paradoxically this is more of a problem with modern HPLCs which have very low volume connections to limit extra-column band spreading than with the old fashioned kind which had a long piece of tube between the injector and column. The sample and mobile phase were thoroughly mixed by the time they got to the column and so the composition of the injection solvent was far less critical.

Peter

I found out, at 75% methanol, the tailing and jagged effects were reduced, but I don't want to use that strong solvent, any suggestions?

By the way, can you explain to me why was the methanol that gave the problem of jagged and tailing peak?

There's a question of definitions here. In reversed-phase chromatography, methanol is a strong solvent, water is a very weak solvent. To rephrase Peter Apps' correct suggestion, your sample should be dissolved in something no stronger than your initial mobile phase.

Thanks everybody, I tried to use weak solvent (less methanol) to dissolve my sample, however my sample can not dissolve well if there is > 20% of water in the solvent mix (Percipitation form). What will happend if I convert methanol to 100% acetonitrile? Will it be batter. With the high % of organic in C18 reverse phase HPLC column, will it cause the column to "die" faster?

The problem is probably the fact that the sample is a vegetable oil. This is causing the problem that you have described as precipitation. Acetonitrile is not going to help, it will create more peak distortion than methanol.

The problem is that all these solvents including your sample matrix are "strong solvents" in RPLC.

You can either inject less, if the analysis is still OK at lower injection volume, or you need to do some sample preparation, or you need to set up your instrument differently to make the injection different, i.e. work with at-column dilution.

The first approach seems to be the simplest, the last one is guaranteed to work with little difficulty, but you will need to replumb your system. Basically, you set up a stream of mobile phase that delivers the methanol, and you inject the sample into this stream. This stream is diluted post-injector and pre-column with second solvent, water and HCOOH.