Macromolecules and other thermolabile biologicals are often buffered and stored in frozen or dried (freeze-dried) state. Crystallization of buffer components in frozen aqueous solutions and the consequent pH shifts were studied in carboxylic (succinic, malic, citric, tartaric add) and amino acid (glycine, histidine) buffers. Aqueous buffer solutions were cooled from room temperature (RT) to -25 degrees C and the pH of the solution was measured as a function of temperature. The thermal behavior of frozen solutions was investigated by differential scanning calorimetry (DSC), and the crystallized phases were identified by X-ray diffractometry (XRD). Based on the solubility of the neutral species of each buffer system over a range of temperatures, it was possible to estimate its degree of supersaturation at the subambient temperature of interest. This enabled us to predict its crystallization propensity in frozen systems. The experimental and the predicted rank orderings were in excellent agreement. The malate buffer system was robust with no evidence of buffer component crystallization. and hence negligible pH shift. In the citrate and tartrate systems, at initial pH < pK(a2), only the most acidic buffer component (neutral form) crystallized on cooling, causing an increase in the freeze-concentrate pH. In glycine buffer solutions, when the initial pH was similar to 3 units < isoelectric pH (pI = 5.9),beta-glycine crystallization caused a small decrease in pH, while a similar effect but in the opposite direction was observed when the initial pH was similar to 3 units > pI. In the histidine buffer system, depending on the initial pH, either histidine or histidine HCl crystallized.