Fig. 1. Optimised separation of a 9-peptide mixture on two MS/BVPE capillary columns (Table 1, entries 1 and 2) prepared in the presence of (A) THF and (B) toluene as microporogen, having comparable hydrodynamic properties. Separation conditions: (A) 0–30% B in 10 min, 17 μl/min, 50 °C, UV 214 nm, inj.: 500 nl, 5 ng each; (B) 0–30% B in 4 min, 19 μl/min, 50 °C, UV 214 nm, inj.: 500 nl, 5 ng each; (1) bradykinin fragment 1–5, (2) vasopressin [Arg⁸], (3) methionine enkephaline, (4) leucine enkephaline, (5) oxytocin, (6) bradykinin, (7) LHRH, (8) bombesin, (9) substance P.

Fig. 2. Optimised separation of dsDNA restriction fragments (pBR322/HaeIII) on two MS/BVPE capillary columns (Table 1, entries 1 and 2) prepared in the presence of (A) THF and (B) toluene as microporogen, having comparable hydrodynamic properties. Separation conditions: (A) 15–30% B in 7 min and 30–35% B in 4 min, 17 μl/min, 50 °C, UV 254 nm, inj.: 500 nl, 50 ng total; (B) 15–40% B in 5 min, 20 μl/min, 50 °C, UV 254 nm, inj.: 500 nl, 50 ng total. Peak labelling denotes the number of base pairs.

Fig. 3. Effect of the column inner diameter of monolithic MS/BVPE supports on the separation efficiency towards proteins: (A) 80 mm × 0.2 mm capillary column, (B) 80 mm × 0.53 mm capillary column, (C) 90 mm × 1.0 mm microbore glass column, (D) 90 mm × 3.0 mm conventional size glass column. Separation conditions: (A) monolith Table 1, entry 3; 15–50% B in 5 min, 10 μl/min, 50 °C, UV 214 nm, inj.: 500 nl, 10 ng each; (B) monolith Table 1, entry 4; 15–50% B in 5 min, 60 μl/min, 50 °C, UV 214 nm, inj.: 500 nl, 30 ng each; (C) monolith Table 1, entry 5, 15–50% B in 5 min, 0.4 ml/min, 50 °C, UV 214 nm, inj.: 5 μl, 100 ng each; (D) monolith Table 1, entry 6, 15–50% B in 4 min, 2 ml/min, 50 °C, UV 214 nm, inj.: 10 μl, 400 ng each.

Fig. 4. Effect of the column inner diameter of monolithic MS/BVPE supports on the separation efficiency towards oligonucleotides: (A) 80 mm × 0.2 mm capillary column, (B) 80 mm × 0.53 mm capillary column, (C) 90 mm × 1.0 mm microbore glass column, (D) 90 mm × 3.0 mm conventional size glass column. Separation conditions: (A) monolith Table 1, entry 3, 0–18% B in 1 min and 18–30% B in 10 min, 10 μl/min, 50 °C, UV 254 nm, inj.: 500 nl, 5 ng total; (B) monolith Table 1, entry 4, 0–18% B in 1 min and 18–30% B in 10 min, 60 μl/min, 50 °C, UV 254 nm, inj.: 500 nl, 20 ng total; (C) monolith Table 1, entry 5, 15–30% B in 10 min, 0.4 ml/min, 50 °C, UV 254 nm, inj.: 5 μl, 100 ng total; (D) monolith Table 1, entry 7, 15–35% B in 11 min, 1.5 ml/min, 50 °C, UV 254 nm, inj.: 10 μl, 400 ng total.

Fig. 5. Run-to-run reproducibility and long-term stability of a monolithic MS/BVPE capillary column (Table 1, entry 1) demonstrated by the separation of protein mixtures; (A) chromatographic run 1 and (B) chromatographic run 100. Separation conditions: 15–50% B in 5 min, 10 μl/min, 50 °C, UV 214 nm, inj.: 500 nl, 20 ng each; (1) ribonuclease A, (2) insulin, (3) cytochrome c, (4) lysozyme, (5) α-lactalbumin, (6) BSA, (7) β-lactoglobulin B, (8) ovalbumin.

Fig. 6. Batch-to-batch reproducibility of monolithic MS/BVPE capillary columns (Table 1, entry 3), determined by the separation of a 5-protein mixture (A–C) and by the separation of oligonucleotides (D–F). Separation conditions: see Table 4.

Recipes for MS/BVPE monoliths considered in this study; AIBN (1%, w/v, with respect to the total volume of the polymerisation mixture) was used as initiator in all cases, polymerisation conditions: 65 °C for 24 h in all cases

Summary of chromatographic parameters extracted from Fig. 5 showing run-to-run reproducibility as well as long-term stability (chromatographic parameters: see Fig. 5)

Porosity data obtained by mercury intrusion porosimetry of three different polymer batches, prepared according to recipe 3 (Table 1) in glass vials

Chromatographic data obtained from the separation of proteins and oligonucleotides on three independently prepared MS/BVPE capillary columns (80 mm × 0.2 mm) (Table 1, entry 3)