Chromatography Forum

Hello,

I am having an issue getting a stable baseline with TEAA (100 mM or 50 mM) as my weaker MP. I have prepared the buffer 4 times now, and it is degassed by membrane filtration, and more recently adding stirring under vacuum for 20 mins (no in-line degasser).
The recipe for the buffer is (100 mM):
water 850 mL
1.0 M TEAA 100 mL
acetonitrile 50 mL
Adjust the pH to 7.0 by addition of TEA

I've noticed the cyclic noise in the baseline, with pressure changes. I changed the plunger seals on the pump, and made sure it is properly primed each time. Now I still have the noise but no pressure fluctuation. If I equilibrate the system with water/methanol the baseline looks normal.

I am new to this companies QC department, but from what they tell me all the reagents are new (less than 1 month), and I am using HPLC grade water and acetonitrile to prepare the buffer. Is it okay to use this purchased 1.0 M TEAA solution or better to use TEA and acetic acid?
Any suggestions would be appreciated.

Is your ACN percentage in your instrument method 5%? If not, you should correct it and have look at your baseline again. Probably it helps.

That is strange-looking noise. Very reproducible and with a period of *exactly* 2 minutes (at Least on the blown-up section). If you cut the flow in half, does the period double?

That is strange-looking noise. Very reproducible and with a period of *exactly* 2 minutes (at Least on the blown-up section). If you cut the flow in half, does the period double?

Yes I changed the flow rate to 0.5 mL/min and the period doubled. Also increasing the flowrate to 1.5 mL/min decreased the period. I also noticed a small pressure fluctuation today when running the instrument which correlates to the increase and decrease of absorbance.

Talking to Waters tech service they told me it was likely the inlet check valve. Took those out and sonicated them in methanol and replaced them. Still the same noise after.

I am working with a Waters 1525 Binary pump, are there any other components I should be looking at? HPLC is not my specialty, I am coming from a synthetic organic background.

Are you mixing your mobile phase (adding organic) on-line or using premixed mobile phase.

If you are mixing on-line, try premixing your mobile phase and put both inlet lines in the same jug. If the problem goes away, that suggests a mixing problem. If it continues, it suggests that one side or the other has a flow problem. In that case, try pumping 100%A for a while and then 100%B (both lines still in the same jug). If you see flow issues on one or the other, that tells you where the problem is. Could still be check valves, or possibly seals, or an electronic (motor problem).