Hi All,

I am working on a CEC-HPLC method development. My problem is this: if I inject 0.5 ug of my peptide in a 50ul inj.vol. by diluting a 50ul volume of 0.1mg/ml solution to 500ul gives different peak area from my previous way of dilution. The previous way is to add 20ul from a 0.1mg/ml solution to 60 ul buffer and inject 20ul to give 0.5ug.
Why I am seeing different peak areas i.e. 5 times difference?

Thanks for any advice, comments!

Ananda

With what do you dilute to 500µL? Note that a small (not 5x!!) discrepancy can arise by diluting TO 500µL or by adding two solutions and assuming that they add up arithmetically (remember: In chemistry 2+2 need not be 4).

I am using the same pH 5.75 buffer for all the sample dilution in both ways. Just now I found out that this difference is seen in our QC lab and when I do it in my R&D lab it is OK. So I assume this is something to do with their HPLC system or column or buffer prep.

Thanks for your time and the comment!

Ananda

Hi Ananda,

Are you/they using an autosampler for injection? If so, check the minimum volume needed in the vial to draw up 50uL.

Hi Sir/Madam,
Well i want to know about the adsorption isotherm in liquid chromatography. Please say something on it.
Bye for now.