On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface

A high performance liquid chromatography system, a sample preparation devic e, and an imaged capillary IEF (CIEF) instrument are integrated and multipl exed on-line. The system is equivalent to two-dimensional polyacrylamide ge l electrophoresis (2-D PAGE), by transferring the principle of 2-D separati on to the capillary format. High performance liquid chromatography (HPLC) p rovides protein separation based on size using a gel filtration chromatogra phy (GFC) column. Each eluted protein is sampled and directed to a novel mi crodialysis hollow fiber membrane device, where simultaneous desalting and carrier ampholyte mixing occurs. The sample is then driven to the separatio n column in an on-line fashion, where CIEF takes place. The fluidic technol ogy used by our 2-D system leads to natural automation. The coupling of the two techniques is simple. This is attributed to high speed and efficiency of the sample preparation device that acts as an interface between the two systems, as well as the speed and simplicity of our whole column absorption imaged CIEF instrument. To demonstrate the feasibility of this approach, t he separation of a mixture of two model proteins is studied. Sample prepara tion and CIEF were complete in just 4-5 min, for each of the eluted protein s. Total analysis time is about 24 min. Three-dimensional data representati ons are constructed. Challenges and methods to further improve our instrume nt are discussed, and the design of an improved horseshoe-shaped sample pre paration - sample loop membrane interface is presented and characterized.