I am performing protein fractionation using RP HPLC. I wanted to do relative abundance of protein present using area % as a measure. But the software is not able to collect accurate peak starting and end point. I want to calculate peak area manually. What is the procedure for that. What peak characteristic data should I have to do it?
Hi Siddarth, you can use old and simple technique: 1. You can use a planimeter (https://en.wikipedia.org/wiki/Planimeter) to measure the area. 2. You can cut out the peaks and weigh them on an analytical balance. These two techniques were used in the era before integrators and computers entered the laboratories. - Markus -
Convert all the peak area given by the software after injection into an excel sheet. Manually integrate your known peaks by deleting others. Sum all the value. Use the following calculation to measure the Area % of individuals.
=Individual Area/Total Area x 100
The method that Markus mentioned above (cutting peaks out with scissors and weighing them) is exactly how I used to determine peak areas when I first started out doing chromatography in the 1980's. All we had were strip-chart recorders, no integrators. It worked very well and does not require any computers, just a good quality balance and sharp scissors. This method still works today and is accurate!
HPLC-software (old or new) should allow manual peak integration. Not ideal, but feasible. Plus, please seek advice from the vendors of that software, they should be able to let you know how to either improve the integration parameters or how to integrate manually...
Any other method (and, yes, I remember scissors next to the HPLC) is completely outdated. Reporting values and results based on the suggested approaches will likely result in quite some "nasty" questions.
My two cents.
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