Chromatography Forum

I have an Agilent HPLC 1200 connected to a DAD as well as a 6430 MS. I use it for HPLC-DAD detection using 50mM phosphate buffer as MP. I also use it for MS in other applications. I wonder if phosphate buffer will stay in the LC system and affect my MS analysis.

Likewise, will the ammonium acetate or formate buffer stay in the LC system to affect UV detection?

Any way to eliminate these buffers completely when I switch between applications?

Please comment.
Thanks in advance.

In both cases, the presence of *very* low levels of the buffers in question are unlikely to cause problems.

The issue with phosphate in MS is volatility, with phosphate building up in the ionization source. If you do thorough flush of your system, the amount of non-volatile CRUD coming from your samples will probably far exceed any traces of phosphate left behind.

The issue with UV is absorbance at low wavelength. Unless you are going *very* low (200 nm or below), even 0.1 mM of acetate or formate probably will not give a significant background over trace junk in your acetonitrile.

If the mobile phases are otherwise compatible, a useful rule of thumb is that it takes about 10x the system volume to get reasonably complete washout. I'm paranoid, so I would probably go longer than that. When switching to the UV detector, you can track the washout of formate or acetate by simply monitoring the signal at 205 or 200 nm.

Thanks for your comment.
I have one more question. Will phosphate, acetate or formate form salts adhering to the interior of tubings or junctions and release itself during LC?

I suppose it's possible, but I would rank it as *very* unlikely to be a problem, if for no other reason than the surface area of tubing and fittings is negligible compared to that inside the pores of a typical column packing. Assuming reversed-phase chromatography, acetate may stick somewhat via hydrophobic interaction, but it will come off very quickly.

One comment: we have a method running at 200 nm and we experienced occasionally some problems with high baseline and noise.

It turned out that the person using the instrument before had used a very UV-absorbing mobile phase. This mobile phase was sitting in on the mobile phase filters in the bottles - which was enough to contaminate the new "UV transparent" mobile phase.

The issue with UV is absorbance at low wavelength. Unless you are going *very* low (200 nm or below), even 0.1 mM of acetate or formate probably will not give a significant background over trace junk in your acetonitrile.

When switching to the UV detector, you can track the washout of formate or acetate by simply monitoring the signal at 205 or 200 nm.

The UV cutoff for acetate and formate is stated to be ~210nm which means if you use these additives at this wavelength the mobile phase will be absorbing a significant percentage of the lamp energy. This limits the linear range of your detector, increases baseline noise and increases the likelihood of detector artifacts like negative peaks.

The UV cutoff for acetate and formate is stated to be ~210nm which means if you use these additives at this wavelength the mobile phase will be absorbing a significant percentage of the lamp energy.

True, but these are typically used at concentrations in the 10 - 100 mM range. The issue is the level at which any residual material bleeds off after the system has been "flushed". That's why you monitor the signal until things stabilize.

One comment: we have a method running at 200 nm and we experienced occasionally some problems with high baseline and noise.

It turned out that the person using the instrument before had used a very UV-absorbing mobile phase. This mobile phase was sitting in on the mobile phase filters in the bottles - which was enough to contaminate the new "UV transparent" mobile phase.

Thanks Mattias. Can you please tell me what the "very UV-absorbing mobile phase" is?

I appreciate all comments from Tom, Mattias and carls.

One comment: we have a method running at 200 nm and we experienced occasionally some problems with high baseline and noise.

It turned out that the person using the instrument before had used a very UV-absorbing mobile phase. This mobile phase was sitting in on the mobile phase filters in the bottles - which was enough to contaminate the new "UV transparent" mobile phase.

Thanks Mattias. Can you please tell me what the "very UV-absorbing mobile phase" is?

I appreciate all comments from Tom, Mattias and carls.

I think it was something with EDTA, which apparantly has a very high UV absorbance.

Thanks a lot for your sharing

True, but these are typically used at concentrations in the 10 - 100 mM range.

This may not be the right thread for this discussion but I often hear people discussing using formate or acetate at 210nm. Even at 10mM the absorbance of this additive is still >0.5AU which increases the noise and number of artifacts. It seems many working with these additives in the low UV range forget they are workng "on top of" the background absorbance. Perhaps it is assumed the "low" concentration of the buffer has reduced the background to negligible values but this is not the case.