Microbore flow-rates and protein chromatography

Reversed-phase chromatography of proteins on microbore columns can achieve sensitivities that exceed those for standard-bore columns by a factor of 10-20, when operated at the same linear velocities. These gains in sensitivity are accompanied by proportional reductions in peak volume. Sensitivities on standard- (4.6 mm I.D.) and narrow-bore (2.1 mm I.D.) columns have been further improved by reducing the flow-rates to those typical for microbore (1 mm I.D.) columns. We have investigated the role of flow-rate in determining peak volumes for a constant time gradient and found that flow-rate affects peak volume to a much greater extent than column diameter. Column length was not found to have a significant effect on either peak volume or sensitivity. We have found that a four-fold reduction in flow-rate results in at least a two-fold reduction in peak volume over the flow-rate range from 25 to 400 microliters/min. Recovery of proteins in smaller volumes should prove beneficial to subsequent protein characterization methodologies.