PS-DVB Monolithic Columns Applied in A Microcolumn Switching Set-up for the Analysis of Peptides and Proteins

Polystyrene-divinylbenzene (PS-DVB) trap columns have been evaluated for microcolumn switching applications. In contrast to traditional stationary phases, which consist of packed particles, the monolithic separation medium is made of a continuous, rigid polymeric rod with a porous structure. The absence of intraparticular void volume increases separation efficiency, allowing for faster separations. Column lifetime is higher compared to packed columns.

Figure 1: Direct injection (b) vs microcolumn switching with monolithic trap column of a cytochrome c digest (a) with 1 pmol injected.

This article demonstrates that the use of monolithic trap columns for preconcentration and desalting of peptides and proteins does not negatively influence chromatographic performance or sample recovery. Sample capacity of the monolithic trap columns (200µm i.d. × 5 mm) is 100 pmol for both peptides and proteins.

Figure 2: Separation of 16 intact proteins (5 ng each injected) on a monolithic capillary column after preconcentration on a monolithic trap column.

Experimental

LC system: UltiMate Plus nano LC system, Switchos

column switching module and FAMOS

autosampler (LC Packings/Dionex)

Trap column: Monolithic trap column, PS-DVB,

200-µm i.d. × 5 mm

Loading solvent: Water with 0.05% hepta fluorobutyric acid

(HFBA)

Loading solvent

flow-rate: 10µL/min

Analytical column: Monolithic capillary column,

PS-DVB, 200µm i.d. × 5 cm

Mobile phases: (a) Water, 0.05% TFA

(b) Water, acetonitrile (50:50%, v/v),

0.04% TFA

Flow-rate: 2.7µL/min

Gradient: 0–70% B in 7 min for peptides

30–100% B in 25 min for proteins

Column

temperature: 60 °C

UV detection: 214 nm; 3 nL flowcell

Direct Injection vs Microcolumn Switching

The influence of the monolithic trap column on chromatographic performance was evaluated by the separation of tryptic peptides of cytochrome c. Figure 1 shows a comparison between direct injection and microcolumn switching with a monolithic trap column. Table 1 lists peak widths at half height (PWHH) for the tryptic peptides with and without preconcentration.

Table 1: PWHH for Tryptic Peptides of Cytochrome c Separated on Monolithic Columns

Protein Separation After Preconcentration Using Monolithic Columns

Figure 2 shows the separation of 16 standard proteins on a monolithic capillary column with sample loading on a monolithic trap column. PWHH were typically between 4 and 8 s.

Conclusion

The experiments demonstrate that monolithic trap columns can be used for preconcentration and desalting of samples consisting of peptides and proteins, without negatively influencing the chromatographic performance or recovery of the compounds. The sample capacity of the monolithic trap columns (200µm i.d. × 5Xmm) is in the range of 100 pmol for both peptides and proteins (data not shown).