Chromatography Forum

I am fresh man, come across the troubel in the process of test nucleotide acid. The sample is standard CMP (cytidylic acid) in phosphate solution. The chromatography condition is followed:
MP: Time(min) A (KH2PO4 25mM,ph 2. B (Methanol ) C (water)
0 2% 0 98%
3 2% 3% 95%
3.10 2% 20% 78%
15 2% 20% 78%
20 2% 0 98%
30 2% 0 98%
Detector:UV(260nm)
Flow rate: 0.8ml/min
Oven Temp: 25℃
Column: Zorbax SB C18 250X4.6mm, AQ

In the test for the first time, the chromatography peak was normal,but in the next day, the sampler was injected, the chromatography peak was very broad, the condition of chromatography is absolutely same to the previous test, even the sampler and MP were not changed either.
In the last, the equilibrium time was extened 20 minitues, in other word, the 30min in last line of time table was modified as 50 min, the peak was normal.
But the peak was very broad again this day, even if the euilibrium time was extened 40 min, the peak was till very broad.
Anyone can tell me what reason it is, how should i solve the problem?
Thanks in advance!

Eluent A, pH of phosphate is 2.8

was the column old?

Could be a dewetting of the column in your initial mobile phase. I suggest to run a blank gradient first, and then inject your sample inot the second gradient. If I am correct, the equilibration time between injections can then be reduced to the original 20 or so minutes.

The column have been used about one month. I have already run the blank gradient before inject sample, the result was still same.
I do not know what is dewetting of the column,would you explain it for me? Thanks a lot!

Dewet means the hydrophobic phase collapses(conventional saying) in 100% aqueous due to the incompatibility between the stationary phase and the aqueous buffer. You should not be the case since your column was designed for 100% aqueous unless the column has short life time under your condition, which I do not have any ideas. Hope some one else will provide some insights. Good luck.