hplc running problem
Posted 03 January 2011 - 01:55 AM
Please,anybody could suggest me the possibilities for my failures.
I am working on the carotenoid's estimation in different tropical fruit varieties..
when I am running the sample through the column(c18), the pressure(4000) is increasing abruptly...after it crosses the maximum(6000), the run is stopping it self.
I tried with different fruit varieties (mango,grape, citrus, orange...), most of them giving the same problem. Especially when I run the column with the Sapota the column stops with in 5-10 minuters as my run should be 20 minutes.
I am expecting the problem may be because of isolation of the carotenoids from sources. I used different compositions of my extraction mixtures..... but no considerable change I observed.
If any of you knows a well practiced protocol and also the causatives of my probelm...... plz suggest me.
Posted 03 January 2011 - 04:02 AM
well, look at this useful link : HPLC troubleshooting guide
you may need to filter your sample before applying, but here you have the link with variety of solutions.
and here is a paper : Comparison of 3 Spectrophotometric Methods for Carotenoid Determination in Frequently Consumed Fruits and Vegetables
All The Good Luck ...
Posted 05 January 2011 - 01:30 AM
I used to filter the solution before ingecting every time.I have gone through the trouble shooting,mostly "Water-organic solvent systems - buffer precipitation.. Solution-Ensure mobile phase compatibility with buffer concentration; decrease ionic strength or water organic solvent ratio." may be helpful to me..
once again thanks..allow me to ask further doubts.
Posted 05 January 2011 - 08:09 AM
will be ready to help as i can ...
hope ur challenge is solved ...
Posted 24 January 2011 - 08:04 PM
if not use a guard column, check the pressure of the guard column and the main columns eparately with the initial gradient of your ethod and if the prssure increases after your sample injection, then disconnect the guard copumn and run the initial gradient thru the column and guard column... if the pressure is high in the guard that means the sample has precipitated in there... then u might have to rethink abt the buffers and the sample compatability!!!
it would be great if u can give in teh details of ur running conditions!!!
hope this helps!!