The Column - September 2008 - (Page 14)

Eeltink et al. The Column September 2008 Separation of Complex Peptide Samples using Optimized Column Technology and 1D-LC Conditions Sebastiaan Eeltink, Bas Dolman, Karsten Dekker, Fraser McLeod and Remco Swart, Dionex Corporation, Amsterdam, The Netherlands. The effects of liquid chromatography (LC) conditions (gradient time and temperature) and column length on peak capacity for the separation of tryptic peptides was studied in reversed-phase gradient elution nano LC. To perform the study, 75 µm i.d. columns up to 50 cm in length were packed with 3 µm silica particulate materials and tested. Increasing the gradient time and column temperature helped to increase peak capacity. A maximum peak capacity of 443 was obtained with 50 cm long columns operating with a 120 min gradient time and a column temperature of 60 °C. Introduction Determination of the proteome and identification of biomarkers is required to monitor dynamic changes in living organisms and predict the onset of an illness.1,2 Different approaches are available for the identification of proteins. One method is called shotgun proteomics, in which proteins are digested, the resulting peptides are separated by high performance LC (HPLC) and identification is performed with tandem mass-spectrometric detection.3 Digestion of proteins may lead to a very large number of peptides. For example, it has been estimated that digestion of a cell lysate may produce up to 500000 peptides. The separation of highly complex peptide samples is one of the major challenges of analytical chemistry. Columns packed with microparticulate silica stationary phases remain the gold standard for peptide separations with nano LC.4,5 This efficacy is due to their analytical properties, Author: Sebastiaan Eeltink E-mail: such as high purity, outstanding mechanical strength and large surface areas yielding columns with high loadability capacity. The capillary format of these columns (75 µm i.d.) ensures high mass sensitivity — typically a factor of 5000 higher than 4.6 mm i.d. columns operating at conventional flow-rates. However, to tackle the complexity of current proteomic samples, new column formats must be developed by increasing the column length to increase efficiency. In addition, LC conditions need to be adjusted accordingly to work effectively with these new formats. This article describes the preparation of long packed columns and their testing in the separation of peptides obtained from a tryptic digest of a six protein mixture by nano LC. The effect of column temperature, gradient time and column length on peak capacity is demonstrated. Finally, run-to-run retention time repeatability when applying long gradient times was determined. 14 Epoxy/Getty Images

Table of Contents for the Digital Edition of The Column - September 2008

The Column - September 2008
Market Trends and Analysis: Water Analysis and Testing
Incognito - Turn On, Tune In, Outsource...
Separation of Complex Peptide Samples Using Optimized Column Technology and 1D-LC Conditions
Diastereoselective Separation of Fenvalerate by Supercritical Fluid Chromatography with Tandem Columns
Q&A - Highly Sensitive

The Column - September 2008