Potassium phosphate buffers, while having several advantages over other solvent systems for many high-performance liquid chromatography (HPLC) applications, suffer from a serious disadvantage, namely a high background absorption in the UV region. This is true for reagent-grade phosphates of every supplier tested. The problem becomes particularly acute when very small amounts of substances requiring high concentrations of buffer for elution are to be separated, a typical example being the separation of nucleoside triphosphates on ion-exchange columns_ Although this absorption is obviously due to an impurity. several methods of purification fail to correct the problem. The only published method' for this purpose involves a Dowex l-X8 purification and a series of time-and material-consuming recrystallizations. We have been successful in cleaning up our phosphate buffers by the use of Chelex-100. a Bio-Rad (Richmond, CA, U.S.A.) chelating carboxylic acid cation exchanger_ Chelex-100 is a styrene-divinylbenzene copolymer containing paired iminodiacetate ions which chelates preferentially transition metals. even in highly concentrated salt solutions. We are routinely passing an 1 M solution of KH,PO, through a column of Chelex-100 (lo&200 or 2O(wOO mesh) at a rate of about 1 ml/min -cm' of bed. This treatment effectively removes most of the UV absorbance from the phosphate. to the extent that a gradient reaching 1 M concentration can be used with full scale absorbance at 254 nm of 0.08 or 0.04 with a baseline shift of less than 5 y0 or 10 %, respectively.
The Chelex-100 is suspended directly in 1 M KH2P0, and the slurry used to pack the column. The eluent is collected after 100 ml have passed through. Over 30 liters of 1 A4 solution can be purified through a 120-ml (250 x 25 mm) column without regeneration of the resin. Thus, the process is very economical even if the resin is discarded after use, but regeneration is possible following the manufacturer's instructions. Fig. 1 represents a comparison of the UV spectra of the 1 M KH2P0, solution before and after Chelex-100 purification. The nature of the material removed by the treatment has not been investigated. It is, however, of interest to note that the impurity accumulates on anion-exchange columns at concentrations lower than 1 M.. Thus, if several runs are performed with gradients reaching up to 0.60 or 0.70 M. a subsequent 1 M elution will slowly remove large amounts of UV absorbing material. For the same reason, even if a concentration of 1 M is not routinely reached OCUI-9673/8l/OOOCLOOOO/SOZ.jO