HPLC Products

Normal

Problem

To be clear, the problem we are describing here is when we run a standard, where we already know the concentration, and the peak heights are all larger than we expect; larger than we would normally get.

The first question is:

Is the void volume peak larger too, or is it its noraml size?

If the void volume peak is larger too, the answer is that for some reason, we injected more sample.

There are several possible reasons for this:

A larger loop was fitted. This only applies to manual injection valves using filled loop injection, and to fixed volume autosamplers, where the injection volume is the loop volume. Clearly if you work on your own and don't suffer from Alzheimers Disease, you would hopefully know if you changed the loop. But in a multi-user lab, it can happen.

Injection Volume on a variable volume autosampler was set too high. This could be a tuping error, or it could be that the wrong method was selected in the data system.

The autosampler tray temperature has just been reduced to 4oC. This reduces the volume of the samples, and hence 20ul contains more sample.

If the void volume peak is normal size, then a normal injection volume has been made, and we are looking for a reason for a higher sample concentration or higher detector response.

Incorrect sample dilution. This error could have come from the weighing (usually the error will be a factor of 10) or from making the sample up in 10ml instead of 100ml etc. If it is a small difference in peak size, suspect that the dilution was made when the lab was hot. Hence less sample solvent will have been used to make up to the mark.

Incorrect wavelength used. This can cause some peaks to be smaller and some to be larger, But it could result in all peaks being larger.

Sample Carryover. This means that the injection contains the current sample plus some of the previous sample. Clearly this is worst and most noticeable after a high concentration sample or standard. If the standards are run first, in order of increasing concentration, check the first unknown sample run after the last (highest) standard. Sample carryover is dealt with using the autosampler was routine.

Incorrect Range setting. eg 1AUFS instead of 2AUFS. This tends to give peaks which are larger by a factor of 2.

Lamp Energy. If a new lamp has just been fitted and the old one was really tired, significantly larger peaks can be expected. To avoid this with a deuterium lamp, change the lamp at 2000 hours.

Changed flow cell path length. Suspect this if the flow cell has been changed. The longer the pathlength, the larger the peaks, the greater the low end sensitivity, and the lower the maximum concentration which can be used.