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Chemistry Forums for Students => Analytical Chemistry Forum => Topic started by: lemonoman on August 08, 2005, 07:48:03 PM

Title: Negative Response in HPLC
Post by: lemonoman on August 08, 2005, 07:48:03 PM
A friend of mine ran an HPLC experiment a little while back, and showed me the results today.  There is a series of peaks, as normal, and then for some reason it dips below the baseline.

This friend has drawn, in Paint, a representation of the chromatogram:

(https://www.chemicalforums.com/proxy.php?request=http%3A%2F%2Fi15.photobucket.com%2Falbums%2Fa391%2Flemonoman%2Fchromatogram1.gif&hash=3be12eafa1cc1747ba6672bf9ff67f9a)

Has anybody here seen anything like that on an HPLC chromatogram?  And if so...did you ever find out the cause?
Title: Re:Negative Response in HPLC
Post by: asqa on August 09, 2005, 12:53:14 AM
Reason is as below. If you ae using .
RI detector: refractive index of the analyte lower than that of the mobile phase
reverse detector polarity to obtain positive peaks
 
UV detector: absorption of the analyte lower than absorption of the mobile
phase
use a mobile phase with lower UV absorption; if recycling solvent, use fresh HPLC grade eluent when the recycled mobile phase starts to affect detection

Vacancy chromatography

A technique in which a mobile phase additive causes a positive detector signal output. When a solute elutes from the column, it dilutes the signal and gives a negative peak or a vacancy. The technique has been most recently applied to single column ion chromatography, in which mobile phases that absorb in the UV region, such as citrate and phthalate buffers, are used. When a nonabsorbing anion elutes, it dilutes the UV absorbing background and causes a negative peak. The detector output leads are usually reversed to make the chromatogram look normal.


 

 
Title: Re:Negative Response in HPLC
Post by: asqa on August 09, 2005, 01:04:28 AM
herewith i attached HPLC trouble shoot guide.
I am sure its helps lot