High-performance liquid chromatography column length designed for submicrogram scale protein isolation.
High-performance liquid chromatography of small amounts of protein was readdressed with respect to current gas-phase sequencing technology. Useful primary sequence information can be obtained from as little as 5-100 pmol of material. This corresponds on a mass level to the nanogram to microgram range where, unfortunately, HPLC columns often give low recoveries depending on the size and surface hydrophobicity of the peptide or protein. It was rationalized in this study that reduced column length could have a beneficial effect on recovery without significant loss of resolution. To demonstrate this, six HPLC columns ranging from 0.2 to 25 cm in length were made and evaluated in terms of protein loading and resolution. Column lengths of less than 1 cm were found to increase recovery of surface hydrophobic proteins without loss of resolution, as shown for a standard protein profile. These columns resolve proteins best when loaded with less than 10 micrograms, with recoveries greater than 90%. All column internal diameters were at least 4.1 mm so that standard HPLC pumps could be used to generate gradients.
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D004798:Enzymes / Q000302:isolation & purification; D008433:Mathematics; D008832:Microchemistry; D011506:Proteins / Q000302:isolation & purification*
D004798:Enzymes; D011506:Proteins