The ProteomeLab PF2D protein fractionation system is a rapid, semi-automated, 2 D-HPLC instrument that uses two different methods to separate plasma serum proteins: ion-exchange chromatography using a wide pH range in the first dimension, and non-porous reverse-phase chromatography in the second dimension. Because this methodology has only very recently been introduced in proteomic laboratories, little is known about the characteristics of PF2D fractionation of human serum proteins. To evaluate the system's application in a clinical laboratory setting, the characteristics of the ion-exchange chromatography-based separation were analyzed. Following fractionation of human serum proteins on a linear pH gradient (ranging from 8.0 to 4.0), each fraction was collected in a cool module of the instrument. Different fractions obtained from the first dimension were then pooled together and loaded on classic 2D gel electrophoresis instrumentation. The different spots obtained were then checked against the Swiss-Prot Data-base. A total of 36 human serum proteins were identified in different PF2D-generated fractions. Some important features of the separation system were observed. Different eluted fractions contained different proteins, thus demonstrating the reliability of the fractionation system. The proteins were also fractionated according to the theoretical isoelectric point (pI). This was consistent with the evidence that the vast majority of immunoglobulins, characterized by an alkaline pI, were not retained by the column and were eluted in the unbound fraction. This outcome also underlines a practical advantage: fractions eluted from pH 8 to pH 4 contained virtually immunoglobulin-depleted serum proteins. This finding supports an immediate use of the PF2D system in a clinical setting, where abundant proteins should be clearly identified to enable evaluation of other less abundant, but potentially relevant, species.