In this study, high-efficiency LC-MS/MS separations of complex proteolytic 
digests are demonstrated using 50 mm, 250 mm, and 1m long 
poly(styrene-co-divinylbenzene) monolithic capillary columns. The 
chromatographic performance of the 50 and 250 mm monoliths was compared at the 
same gradient steepness for gradient durations between 5 and 150 min. The 
maximum peak capacity of 400 obtained with a 50mm column, increased to 485 when 
using the 250 mm long column and scaling the gradient duration according column 
length. With a 5-fold increase in column length only a 20% increase in peak 
capacity was observed, which could be explained by the larger macropore size of 
the 250 mm long monolith. When taking into account the total analysis time, 
including the dwell time, gradient time and column equilibration time, the 50mm 
long monolith yielded better peptide separations than the 250 mm long monolithic 
column for gradient times below 80 min (n(c)=370). For more demanding separation 
the 250 mm long monolith provided the highest peak production rate and 
consequently higher sequence coverage. For the analysis of a proteolytic digest 
of Escherichia coli proteins a monolithic capillary column of 1m in length was 
used, yielding a peak capacity of 1038 when applying a 600 min gradient.