Chromatography Forum

Dear All,

I'm running a simple HPLC isocratic method consisting of Ammonium acetate (1 gm/lit) adjusted to pH 5.6 with glacial acetic acid and 100% Acetonitrile as Mobile phase B.The mobile phase composition is 94 % A(Buffer) and 6 % B( Acetonitrile). The monitoring wavelength is 230 nm.
I found a strange problem- when I used a premix solution of the above mobile phase the baseline is very stable but when I mix it through my quaternary pump I found the baseline very unstable. I thought this is a mixing problem hence I tried optimizing the mixing by adding some static mixer of 700 ul but it didn't solved my problem.
Please share if anyone else has faced similar problem and rectified it. Please provide me your inputs.

What is your flow rate and column Dimension?
Do you run a low pressure or high pressure Gradient System?

Acetate shows significant absorption at 230nm, that's why any inconsistencies of the mobile phase clearly show up. You said you have a quaternary pump, so I'd assume this is a low-pressure mixing system? These systems tend to give lower blending accuracy at the extremes of eluent ratios - and you're mixing 94:6, so probably there are little fluctuations of the actual eluent composition. If you mix 50:50 on the very same system, the baseline might look more stable.
Anyway, this is a good example of why I usually advocate premixing .

Acetate shows significant absorption at 230nm, that's why any inconsistencies of the mobile phase clearly show up. You said you have a quaternary pump, so I'd assume this is a low-pressure mixing system? These systems tend to give lower blending accuracy at the extremes of eluent ratios - and you're mixing 94:6, so probably there are little fluctuations of the actual eluent composition. If you mix 50:50 on the very same system, the baseline might look more stable.
Anyway, this is a good example of why I usually advocate premixing .

Thank you very much for your inputs.

What is your flow rate and column Dimension?
Do you run a low pressure or high pressure Gradient System?

The flow rate is 1.2 ml/min and i'm using a low pressure gradient system.