Chromatography Forum

Dear All

I am running a binary gradient (a buffer sol +methanol) program on my knauer hplc, problem is that I having decreased retention time. I thought it is the problem of inconsistent gradient mixing so i replaced the mixing chamber by a union but the problem remains the same. I have the following system.
Knauer pump s 1000
knauer photodiode array detector 2800
autosampler 3900

Somebody please give me a hint to troubleshoot the problem.
afzal123_ahmed@yahoo.com

With my experience, this problem is often related to your substance's property. Is it base ? And your mobile phase pH? Column?

The problem can be many things. The way to discover it is to go step by step through the whole process of analysis starting from the reagents that you've been using and ending with the starting of the sequence. I don't know if you have changed some of the method settings compared to the last settings that you've used (because you need to have previous analysis for comparison, to be able to say that your Rt has decreased)? Also, you should keep in mind that if you by any chance have changed the methanol manufacturer, you can have problems considering Rt due to its purity and properties?

However, the answer to your problem is not quite simple due to the many factors that need to be looked into. Therefore my only advice is to go step by step and revise everything that you've done and the reagents that you've been using, and I'm sure you will find the cause of it.

There is a "step-by-step" guide to troubleshooting on our web site that may help:
http://www.lcresources.com/resources/TSWiz