Optimization of NanoLC System for Nano Columns
The lower the LC flow rate and the smaller the LC column particles, the more essential it is to optimize the LC system to get the best performance from a premium nano-capillary column. At nanoliter flow rates, optimization of all system volumes (especially poorly swept dead volumes) is critical to maximize the performance of premium nanoLC columns. The two LCMS chromatograms below show the separation of a 100 femtomole BSA tryptic digest on the same Premier NanoLC column (75u ID by 150mm long 3u 200A Magic C18AQ) at a flow rate of 300 nanoliters/minute. Figure 1 shows the results of a run on an older nanoLC system that was not optimized for use with the column, while Figure 2 shows the results of a run on a newer system that was carefully optimized for use with the column. The major differences were as follows:
Figure 1 Figure 2
Gradient Delay Volume 5000nl 500nl
Poorly Swept Dead Volume 20nl 0nl
In order to get the best possible performance from a premium nano-capillary column, great care must be utilized to insure optimum performance. Although gradient delay volume (volume from the LC mixer to the head of the column) needs to be minimized to prevent long delays between the start of the run and the elution of the first sample component (as shown in Figure 1 above), the more important volumes to control are any unswept dead volumes in the LCMS setup. These unswept dead volumes can arise from poor connections between the injector and the column and between the column and the ESI emitter. When using a trap column to load samples, great care must also be taken to insure the trap column is properly connected and the chemistry of the trap column and the analytical column are well matched to prevent any unwanted band spreading and/or sample losses.