The demand for separation techniques for intact proteins is increasing with the introduction of a new generation of high resolution mass spectrometers which are able to measure the mass of small to medium size proteins very accurately. Liquid chromatography is a valuable tool for separating these proteins prior to the MS analysis. Intact protein chromatography is most commonly used in a top-down approach in proteomics and to determine expression levels during recombinant protein synthesis.
The size of the protein molecule results in very low diffusion coefficients and therefore slow mass transfer in and out of the pore system. A sufficiently large pore diameter is required to minimise the effects of restricted pore diffusion. We show examples of the separation of intact proteins on a column packed with 3 µm C8 silica with 1000 Å pore size. The molecular weight of the protein examples reported here cover ribosomal proteins (<40 kDa), monoclonal antibodies (~150 kDa) and intact membrane proteins derived from mouse live.