Chromatography Forum

Hi. I'm pretty new in HPLC. I'm assigned to separate 2 DNA fragments (300bp and 2500bp) using HPLC.

Recently I encounter a problem whereby my retention time shortens by about 7 minutes. Not only that, initially there were 2 peaks coming out (at 24 and 29 min) but, with the same sample, same gradient, pressure and flow rate, now the peaks overlapped and come out at 17.5 min.

What could be the reason for this abrupt change in retention time? What should I do to separate the peaks?

First things to check: does the pump work properly? Have I made the solvents correctly?

Flow rate and pressure are fine. And the solvents are freshly prepared and are the same with that used in previous successful runs.

Next questions: what are the solvents? I assume you are running a gradient, please describe... What's the temperature? Etc....

Column: Waters Gen-Pak FAX (4.6 x 100mm) (an anion exchange column)
Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0
Buffer B: 25 mM Tris-HCl, 1 mM EDTA, 1.0 M NaCl, pH 8.0
Flow rate: 0.5 ml/min
Gradient: Load at 100% A. Then 0% to 50 % Buffer B in 1 minute. Then 50% to 60% Buffer B in 30 minutes.


I don't have a temperature controller. But, because my lab ambient temperature only range from 30-35 celcius. I believe this will not affect the HPLC much.