extra peaks in phosphate buffer

[bobbygau]

I am suffering in the extra peaks brought from phosphate buffer in my HPLC analysis. the reagents used for the preparation are both ACS grade not HPLC grade which are much much more expensive. does anyone know the difference between these two grades? will it help if I replace with HPLC grade?

[Andy Alpert]

ACS-grade phosphate contains some heavy metals that HPLC-grade does not. These accumulate on some columns during equilibration and then elute in well-defined peaks during the gradient. You can get rid of them by preparing a solution of the ACS-grade reagent and passing it through a column packed with a low-pressure mixed-bed ion-exchange resin. Alternatively, put the resin beads in a fine mesh bag and swirl it through the solution. There's a detailed procedure for this by Mant and Hodges in Methods in Enzymology (@ 1996 or so). If you want the exact reference, I can cite it on Monday when I get back to the lab.

Having said that, it might be easier and perhaps cheaper just to buy HPLC-grade phosphate reagent.

[danko]

It’s far from certain the phosphate salt is the culprit here. It might as well be the ACN and indeed the water.
I’ve seen all the variations of the above and in addition to that contaminations stemming from the mobile phase containers, filters (if the eluents are filtered) etc.
My point here is the need for identifying the source of the trouble and address it properly.
Best Regards

[Gerhard Kratz]

I fully agree with Andy.
My experience is that even USP standards are not 100% pure and HPLC grade buffers can be qualified against ACS and USP standards. That saves some money and makes your life easier.