Chromatography Forum

Dear all,

I keep fighting with tetracyclines;
I managed to set up a method which works perfectly well on a Waters 2695 and Quattro Premier. The column is a from Waters a SunfireC 18 3.5 um (2.1x 150mm) and the mobile phase is water and methanol with 0.1% of formic acid. In this instrument I have no problem with the Rt of the peak nor with the peak shape. However, when I set up this method on a HPLC 1100 from Agilent and a MS API 2000 I am not able to get reproducibility. I though it may be due to sensitivity but I am not able to reproduce the same peak shape or signal intensity by increasing the sample volume. The Rt and peak shapes move a lot more than one min. The flow rate is 0.250 ml/min and the injection volume is 25 ul.
I will appreciate any idea or suggestion,
Thanks in advance,
Carol,

Carol,

1. What is the sample dissolved in ?

2. What is the initial mobile phase composition ?

JMB

Hi,

The sample is disolve in 90 % of water and 10% of metanol. The gradient starts with 100% of water.

Thanks,
Carol

1. The major difference between good and bad results would seem to be,
Agilent 1100 plumbing vs. Waters 2695 plumbing.

The Agilent system will have a different dead volume, and different mixing chambers. This may be enough to shift the RT's, although I would still expect reproducibility AS LONG AS THE COLUMN IS COMPLETELY EQUILIBRATED FOR EACH INJECTION.

You might increase the time or volume of mobile phase at initial conditions, and then check for reproducibility.

2. I would also check reproducibility & peak shapes on the Agilent system without the MS system connected. Perhaps the LC-MS interface adsorbs/desorbs your analytes and disturbs the peak shapes.

As an aside, I was always told to avoid 100% aqueous phase due to potential or actual "phase collapse" when using C18 columns. However, you do get acceptable results with the Waters HPLC system, so perhaps it is not an issue here.

JMB

Thanks for the reply,
The system apparently works well with other method, I don't know how to check the HPLC without the MS, I do not have any other systems.
Certainly, it looks like the column it is not completely equilibrated, so I will increase a couple of minutes at the beginning and the end of the gradient to see if the chromatography improve. I can also try to start at 95% of Water, that will certainly help with the pressure problems I have some times.
Thanks again,

I will keep you inform,

Carol