Chromatography Forum

Hello All,

I have question regarding using buffers during a gradient run.

These are the current parameters that I run for my method:

A: 0.1% phosphate buffer in H2O
B: 0.1% phosphate buffer in Acetonitrile.

starting conditions of 10%A/90%B increasing up to 10%A/90%B over 6 mins.

All of my compounds are eluting but I am getting a ton of baseline drift. On average 15-20 mAU.

Any thoughts on what I can do to help minimize this? Does it make sense to be treating the Acetonitrile with the buffer?

Also what kind of harmful effects can result from storing my column in 100% solvent B for long term?

Sorry if this is a bit jumbled but thank you!

B

There's a lot of missing information there; things like:
what wavelength?
what counterion?
what temperature?
what pH?

Does it make sense to be treating the Acetonitrile with the buffer?

0.1% is 1 g/L ; depending on the salt you used, that would be something under 10 mM, which is actually fairly low for an aqueous phosphate buffer. I generally don't put buffer in the organic, if for no other reason than adjusting the pH is problematic.

If your drift is non-linear, you may be seeing the effect of refractive index changes during the gradient. Check out the first three minutes of so of this quick tutorial on gradient baseline problems:
viewtopic.php?f=31&t=19085
If your detector flow cell is out of alignment, that may exacerbate RI issues.

Also what kind of harmful effects can result from storing my column in 100% solvent B for long term?

Pretty much none. In fact, storing is 100% organic is commonly recommended unless the column vendor specifies something else for that particular column.

Phosphate buffer in ACN you should avoid. How often do you maintenance your pump head?

wavelength is 275nm
counterion is phosphate
temperature is 40C
pH is 2.5-3


I usually do not use buffers at all and my LC is only 6 months old so I have not performed any maintenance on the pump head. I have not noticed any difference in pressure or anything that would indicate the pump head is malfunctioning. All of my other chromatograms have been fine except until I introduced these buffers.

counterion is phosphate

The counterion to a phosphate buffer can't be phosphate itself; it has to be a cation (sodium? potassium? ammonium? or at that pH, did you just use phosphoric acid -- in which case it's not really a buffer?).

If you just used 0.1% phosphoric acid, then solubility in ACN is less of a concern, and pH measurement is irrelevant.

With detection at 275 nm, it's unlikely that your drift is coming from UV absorbance mismatch (that is much more common at short wavelength).