Chromatography Forum

HI,
I have a problem with retention time.
my gradient is:

Time MeCN buffer phosfate pH 6,5
0 5 95
2 5 95
16 20 80
18 20 80
25 70 30
30 70 30
32 5 95
37 5 95

Some times I have shift also positive or negative of 1 or 2 minutes.
Some day for three four times is all ok after the HPLC didn't work correctly
The molecules is aromatic amines like 2,2'-({4-[(2-hydroxyethyl)amino]-3-nitrophenyl}imino)diethanol.

My HPLC is a Agilent 1200 with quaternary pump e DAD
Thanks for the help

You might have equilibration problem. I don’t know your column dimensions but 5 minutes is not enough to equilibrate column. For 150 mm column I would recommend at least 10 minutes equilibration and for 250 mm at least 15 minutes.

Also make sure that your phosphate buffer is not crushing out at 70% ACN.

Solubility problems can often first appear as deposits on the checkvalves, leading to irregular flow. Monitor the pressure as an additional data channel, and see if that is also varying between runs.

altough it also depends upon the concentration of your buffer, it is still better to use pre-mixed solutions, because altough at the end of the mixing chamber you have the right composition, it is not true at the level of the proportionning valves and you might be inducing salt deposits especially on the check valves which looks like your problem as Mark Tracy said.

what is your washing protocol when you finish your work?

Thanks at all,
Now I try to increase my time of equilibration to 15 minutes because my colum is a 250mm and I hope that the problem is this.

Thank you for your help

HI,
I have try to increase my time of equilibration and also I have increase the temperature to 40°C but this morning I have done three injection and either chromatogram has retention times different. I don't understand. The colum is a Ascentis RP amide of 250mm and the molecules that I have to separate are: 4-amino-3-nitrophenol; 2-{[2-(2-hydroxyethoxy)-4-nitrophenyl]amino}ethanol; 2,2'-({4-[(2-hydroxyethyl)amino]-3-nitrophenyl}imino)diethanol; 4-[(2-hydroxyethyl)amino]-3-nitrophenol; 3-[(4-amino-6-bromo-1-hydroxy-8-imino-5-oxo-5,8-dihydronaphthalen-2-yl)amino]-N,N,N-trimethylanilinium.

was the change going one way or was it sporadic and going back and forth.

if it was going back and forth it could be your check valves.
what software do you use? have you monitored the pressure like Mark tracy suggested?

Dear Tonja

I agree with all other experts view but I have one suggestion .

Please call service engineer of Agilent & ask him to check Board of Pump whether any malfunctioning is taking place electronically .

Also ask him to check Composition of Pump which is most of the time probable reason for Retention time shifting .