Investigation and optimization of the extra-column band broadening in micro-flow capillary liquid chromatography

We report on the examination of extra-column band broadening (ECBB) effects as they can be observed when using commercially available HPLC products for micro-LC, with the aim of proposing some general rules for a rational design of the instrument set-up. For this purpose, we systematically assessed the ECBB contribution of the different LC instrument parts under fixed isocratic measurement conditions, using coumarin compounds with a retention factor (k) of respectively ∼1.2 and ∼2.6 at a flow rate (F) of 2.0 μL/min and a commercial LC column with an inner diameter (i.d.) of 0.2 mm. To avoid that the measurement itself would affect the ECBB, detection was carried out using an on-capillary LED induced fluorescence detector. With this approach, the ECBB effect of (1) the flow-channel tubing i.d., (2) the tubing union, (3) the connection fitting, and (4) the injection valve was quantified in terms of its volumetric peak variance. Results show that the ECBB of a standard instrument set-up can be reduced with hundreds of nL² per optimised extra-column instrument part. For instance, the use of the commercially available tubing with unified ferrule-nut structure, which has become very popular because of its user- friendliness, causes an additional peak variance (Δσ_v²) of ∼300 nL² compared to that of carefully manually-prepared tubing connections (with the same 20-25 μm i.d.) using conventional ferrules, nuts, and sleeves. To emphasize the importance of a proper ECBB control in practical LC analysis, we also investigated the impact of the post-column tubing i.d. for the gradient separation of peptides (cytochrome c digest). The ECBB effect of the post-column tubing i.d. was found to be larger than in the isocratic, small molecule case, because the combination of the well-compressed peaks in the LC column and the stronger ECBB effects caused by the slower diffusivity (D_m) of peptides compared to that of small-sized molecules (σ_v² ∝ 1/D_m) makes such separations very vulnerable to ECBB performance losses.