Chromatography Forum

I want to use a buffer at pH 5.2 that is UV transparent. This rules out Phosphate so was considering Citrate.

I've heard that citrate attackes stainless steel, Is this still the case with modern equipment ?

Hi Stanlee,

Actually it’s the other way around – phosphate is UV transparent and citrate isn’t that much.
But it all depends on the detection wavelength. If you’re planning to detect at short wavelengths (e.g. 210 – 220), you’re in trouble with the citrate buffer.
On the other hand phosphate does not have any buffer capacity at the desired pH.

Best Regards

As far as I am aware, your citrate buffer is also going to attack the stainless steel tubing in its path as well.

If you plan to use this, then you need to determine all of the wetted stainless tubing on the HPLC you are using. If the assay is routinely run, I would change out all the wetted tubing every 6 mo to year to help avoid any issues.

If you are going to use citrate you might as well try acetate...less worry about the steel.

What about using 10 mM Triethylammonium Acetate (TEAA)? It has a buffer range of 3.8 - 5.8 for the first dissociation.

Make it by adding about 0.7 mL of TEA and 0.6 mL of acetic acid to 400 mL of water. Adjust to the desired pH using additional TEA or acetic acid. Dilute to a final volume of 1 L with water.

Dunno if this is going to work for the UV range you desire though...

Shaun

Be careful when using TEAA: it is an ion-pairing reagent that will alter the characteristics of RP stationary phases. The quality of TEA is also critical. I run into problems in the past with TEA (contaminants). You might have to try different brands and purities.
I would try a regular sodium acetate or citrate buffer. We are using citrate buffer quite extensively (0.02mol/L, pH=6) and haven't experienced any problems. The pumps are stainless as are the switching valves. The tubing however is PEEK.

Citrate has chelating effect. If this is the buffer you plan to use for a long time, you might consider using a non-metallic (PEEK) LC system. As mentioned above, if you use UV detection, citrate buffer is not compatible with low wavelengths.

Wouldn't be Ammonium Acetate buffer, adjusted with acetic acid (or ammonia solution) the buffer of choice in this situation? There should be no significant problem at the 200-220nm wavelength range, and is a volatile buffer, suitable for mass spec, but without ion-pair effect.

Regarding acetate buffers,

It is true that acetic acid hasn't absorption maximum in that range (in aqueous solution max. absorbance is at 247nm), but it still extensively absorbs in the range between 200 and 220 nm. One could try this buffer, but I think that isn't best solution due to the great background absorption in that wavelength range.

Regards