Liquid Chromatography

[chsp]

Sir,

I am working in the field of HPLC method development.

I would like to know

why the baseline in the gradient runs in the region of 210 to 250 nm drifts towards negative (drift is about -100 mau), while I am using acetonitrile and volatile buffers like ammonium acetate, ammonium formate, formic acid, trifluoroacetic acid etc.?

Kindly give your suggestions.

[Alex Buske]

CHSP,

I guess your solvent mixture gets less absortive.
Without some more information it is not possible to be more precise.

Alex

[Provetech]

I agree with Alex, absorbance of mobile phase changes with the composition of its. For example, if a 50-50 mixture of ACN-water has 0 for absorbance and ACN is less absorbtive, if your gradient increases in water the total aborsbance of the MP is weaker than at the start of the run, the absorbance decreaces and the baseline becomes negative.... (f.e abs is -0.2)

That's my opinion.

[tom jupille]

Also, the refractive index changes (which will have some effect on most UV detectors). Generally, RI effects are non-linear and more noticeable at longer wavelengths.

[JA]

chsp,
The buffers you've listed are all carbonyl-containing and are known to absorb at low-UV wavelengths where you are working. As Provetech mentioned, the absorbance of your MP at the end of the gradient is less than the initial conditions due, mostly, to the decrease in concentration of your buffer additive.

Using an additive in line A and an organic solvent in line B is a rough and ready means of buffering a system. If you have a multi channel low pressure mixing system you can minimise the baseline deflection when using salts by utilising 3 channels, for eg. A=H2O, B=ACN and C=aqueous buffer. Modify your gradient to draw a constant 10% of line C and form a gradient using A and B. Prepare your buffer at 10x the desired concentration (eg. 0.1 M). This idea is reproduced from many Waters application notes I've seen.

If you're just using the acids, you can add them directly to both the Water and Acetonitrile.