Chromatography Forum

Hello everybody,

I just like to know if there is of any importance if I add the internal standard when I prepare the mix (methanol, borate buffer, derivatization mix, serum) or I can add it aswell afterwards when I dilute the above mentioned mix with mobile phase?

I am asking this because I ran 2 samples, same dillution, one with serum from patient containing the amino acid I was looking for the other without, in the first one I added the internal standard at the same time when I made the dillution in the other one I added it in the mix and I obtained two very different amplitudes for the internal standard (homoserine) first time 150 mv second time 900 mV.

Please let me know what should I do or what should I change.

Thank you in advance

I see a difference in chemistry in what you describe. If you add the internal standard with derivitization reagent and then dilute or dilute the mix with derivitization reagent and then add internal standard, the quantitiy of internal standard derivatized may change. From what I read, however, it appears that the difference between the samples is that one has serum and has been diluted and the other does not have serum and was diluted? If you have more than one thing changing at a time, you can not tell which of the things that are changing are likely to be related to the problem.

Thank you for your answer Mr. Hilton. In fact in both cases there is serum, the difference is that one serum contains the acid we look for the other one does not contain the acid. So I am talking about two different serum samples.
I am sorry that I did not express myself right the first time. So in this case would you consider normal to have a low amplitude internal standard peak when I add the internal standard at the same time with the mobile phase?

Thank you

I would suggest that you try addign the internal standard at the same point in all samples and see what happens. Without knowing the details of what you are doing, such as which derivatization reagent you are using or the degree of dilution, I have no idea what to expect.

Hi,

What I am trying to determine is the concentration of tranexamic acid in the samples. I use the ortho-phthalaldehyde (OPA) precolumn derivatization. As internal standard I use homoserine at a final concentration of 0.25 mM.

Thank you for your recomandations

I expect the rate of shiff base formation to be concentration dependant and probably pH dependant. Dilution would slow the rate of reaction, so time becomes an important factor as well.