Preparing a calibration curve is a crucial step. In this step, you will be able to have a full image concerning your chromatograms and will determine whether to continue the same validation method or will modify it.

In this simple article, I will represent a simple solution for additional peaks that appear in the first and after the second injection in HPLC.

Please note: all figures in the article were prepared using HPLC Simulator for educational purposes only and not for publishing nor experimental purposes.

 

First injection:

Mostly the peaks from the first run are associated with isocratic elution, for example in figure (1) is representing acetanilide using 100% Methanol.

Figure (1)

This is an ideal peak shape that would fit the calibration curves, and the area of the peak must increase while increasing the concentration.

 

In some situations, in the first sample run, additional peak might appear. The additional peak itself is not the issue rather than its retention time (RT) because this might result in masking the main peak or interfered with other peaks especially if we were using an internal standard (IS) or detecting multiple peaks in the same sample.

This scenario is displayed in figure (2) when analyzing acetanilide and propiophenone using pure methanol 100%. In this situation, we can change the RT by changing the solvent fraction (% v/v).

Figure (2)

 

When changing the solvent fraction, methanol: water ( 9:1), we will notice increasing of RT of propiophenone and the peaks will start to separate figure (3). Since the chromatogram will show two peaks but it is not a convenient solution because the peaks should be completely separated.

Figure (3)

 

Decreasing the methanol proportion to 75%, methanol: water ( 7.5:2.5) will lead to a complete separation of the peaks figure (4).

Figure (4)

Second injection:

Assuming you had a single or double separated peaks in your first sample run and RT was not an issue. You started to inject the second sample but noticed an additional peak that did not exist in the first run.

This peak most likely came from the first sample run but its RT was insufficient to appear on the first but so that it will appear in the second injection.

To solve this issue you have to determine the total RT for this additional peak and decide whether you will elongate the total RT or will decrease it.

 Total RT for additional peak= first run time+additional peak RT in the second run

 

For example:

In the first injection, while everything was fine, the total run for HPLC was 33 minutes and RT for the main peak was 23 Min, RT=23 Min.

For the second sample run, we noticed the additional peak on 24min< you will have a bigger peak or double head peak due to peaks interactions>

That means the additional peak requires total run for 57 min to appear.

So, there will be three solutions:

1.        You change the total run from 33 min to 57 Min

This will guarantee you that you will have your main peak, and the additional peak separated because it will appear at the end of your run. But this method will consume lots of time and it is not recommended specially if you were using manual injector HPLC.

2.      You decrease the current RT that will shift the additional peak to the right

If the RT for your main peak was 23 min, so you can change total run from 33 min to 31 min.

This will lead the extra peak to appear at RT= 57 min – 31 min =26 min

This means you will have your main peak at RT = 23 min and the additional peak at RT= 2 min so you separated them.

3.      You increase the current RT that will shift the additional peak to the left

If the RT for your main peak was 23 min, so you can change total run from 33 min to 40 min.

This will lead the extra peak to appear at RT= 57 min – 40 min =17 min

This means you will have your main peak at RT = 23 min and the additional peak at RT= 17 min, so you separated them.

I hope this simple article will aid you to chose the optimum solvent fraction (% v/v) and total run time for your analytical method.

 

Mohamed Abouzid

Published by Mohamed Abouzid