**2.4 Monolithic silica columns**

Special features of monolithic silica columns circumstanced the successful application in proteomics. In contrast to conventional particle-packed columns, monolithic silica columns are made of a continuous piece of porous silica, utilizing a sol-gel process leading to rod columns, which possess a defined bimodal pore structure with macro and meso pores in the micro- and nanometer range (see Figure 5).

Fig. 5. SEM picture of a cross section from a silica monolith. Total porosity > 80%. The mesopores form the fine porous structure (average pore size 13 nm) and create the large uniform surface area on which adsorption takes place, thereby enabling high performance chromatographic separation. The macropores allow rapid flow of the mobile phase at low pressure. Their average size is 2 μm.

injection to remove the solid contaminants and precipitations. Even so, some components tend to agglomerate/precipitate with the time while samples queue up in autosampler. Therefore, an additional in-line filter is highly recommended. One should keep in mind that the operational flow rate has enormous impact on the molecular size distribution when employing a RAM column. Higher flow rates can shift the molecular range of the trapped molecules to lower values as smaller molecules need less time to penetrate the pores. Also higher flow rates could alter the hydrodynamic volume of the biomolecules. Higher molecular mass molecules will be enriched operating at lower flow rates. Column temperature affects the viscosity of the mobile phase and, consequently, the diffusion ratio and influences the speed of mass transfer. Carefully performed optimizations of the

Special features of monolithic silica columns circumstanced the successful application in proteomics. In contrast to conventional particle-packed columns, monolithic silica columns are made of a continuous piece of porous silica, utilizing a sol-gel process leading to rod columns, which possess a defined bimodal pore structure with macro and meso pores in the

Fig. 5. SEM picture of a cross section from a silica monolith. Total porosity > 80%. The mesopores form the fine porous structure (average pore size 13 nm) and create the large uniform surface area on which adsorption takes place, thereby enabling high performance chromatographic separation. The macropores allow rapid flow of the mobile phase at low

chromatographic parameters ensure the success of the analysis.

**2.4 Monolithic silica columns** 

micro- and nanometer range (see Figure 5).

pressure. Their average size is 2 μm.

One of the major of those special features is low column backpressure. Low backpressure is not only nice to have, but a must, as setting up multidimensional separation platform for proteomics it allows one to select a desired flow-rate from a broader range (see Figure 6).

Fig. 6. Estimation of monolithic silica columns flow rates and mass loadability per column. Columns 2, 3 and 4.6 mm I.D. are 10 cm long; columns 50 µm, 100 µm and 200 µm are 15 cm long.

Prof. Regnier group, demonstrated the advantage of flow rate variation possibility for 4.6 mm I.D. Chromolith Performance column. It was concluded that silica monolith reversedphase chromatography columns show little loss in the resolution of peptides ranging up to several thousand in molecular weight as mobile phase velocity is elevated from the conventional 2.5–25 mm/s (Xiong et al., 2004). Moreover, at 25 mm/s with a 100 mm length column, operating pressure did not exceed 150 bar. This is well within the pressure limit of most commercial LC instruments. The separation of a tryptic digest of cytochrome *C* in 6 and 60 min seemed almost identical. Resolution at 25 mm/s linear velocity was 77% of that at 2.5 mm/s. It was conluded that the fact that peptide separations could be achieved 10 times faster than with a conventional packed column with moderate loss in resolution could have a major impact on analytical throughput in proteomics.

Combination of different sizes fulfils the injection volume requirement for various samples. The possibility of being able to vary the flow-rate over a large area up to very high linear flow velocities combined with the robustness of the monoliths also reduce considerably the "down times" during washing and re-equilibration of the column (Rieuxet al., 2005).

Important to notice, that comparing a particulate and a silica monolithic guard column showed that the particulate column was clogging much faster than the monolithic column (Machtejevas et al., 2007). 120 injections of plasma (50 µl each injection) led to an increase of approximately 6 bar at the particulate column back pressure, while at the monolithic column the back pressure rise was only approximately 1 bar. The life time of the short silica monolithic columns used as a trap column or as a guard column heavily depends on a type

Profiling of Endogenous Peptides by Multidimensional Liquid Chromatography 239

(Kele & Guiochon, 2002) investigated the reproducibility of the preparation of the first columns getting reproducibilities higher than with particle based columns. Because of their capacity to perform fast separations they can be used for fast screening methods and applications in multidimensional chromatography systems. Conditioning and regeneration of these monolithic columns can be done in a short time when compared with the corresponding capillary packed columns, thus making more effective use of costly LC-MS equipment. They can be easily integrated in fully automated systems to perform unattended runs. These columns are flexible and they show a good performance at both low (1.5 µl/min) and high (4.5 µl/min) flow rates. Such flow rate range is highly compatible with MALDI plate spotting strategy. Fraction could be spotted directly, then the flow of 4 µl/min allows to spot up to 8 fractions. If the flow rate is set to 3 μl/min, and an equal flow of MALDI matrix solution is added post-column (7 mg/ml re-crystallized αcyanohydroxycinnamic acid, 2 mg/ml ammonium phosphate, 0.1% trifluoroacetic acid, 80% acetonitrile) and the combined eluant is automatically spotted onto a stainless steel MALDI target plate every 6 s (0.6 μl/spot), a total of 370 spots obtained per original SCX fraction (Fort et al., 2009). Haffey demonstrated similar approach and obtained 3828 MALDI-TOF spots from the 12 SCX fractions (Haffey et al., 2009). Such a separation strategy offers

The analysis concept is based on an on-line sample preparation and a two-dimensional LC (see Figure 7) system: pre-separating the majority of the matrix components from the analytes which are retained on a RAM-SCX (LiChrospher 60 XDS (SO3/Diol), two 25 x 4mm

**RAM-SCX** W

Fig. 7. Multidimensional chromatographic separation platform with integrated on-line

**C-18 C-18**

**SCX**

**MS-MS**

enormous discrimination power and imposing peak capacity.

HP 1100 Degasser

HP 1100 Bin Pump

**3. Application example: The case study** 

W

HP 1100 DAD detector

sample clean-up.

HP 1100 Autosampler

HP 1100 Degasser

HP 1100 Bin Pump

and a volume of the bio-fluid injected. After injecting plasma column performance dropped drastically when the volume of half column volume was injected. For urine the column stability was at least 20 times higher. This is definitely related to the sample complexity. Comparing the life time of the same dimension of monolithic silica columns and particle packed columns under same conditions; monolithic silica column life time was at least double compared with particulate packed column of the similar dimension. This is not a surprise, as any particle packed column contains particles and frits to maintain particles in the column. The flow through between particles is much smaller that the particle size itself, for example, if the column packed with 2 µm particles the space between particles is about 0.5 µm. Monolithic silica column made as one single spongy rod, does not contains frits, and flow through pores (macropores) are about 2 µm diameter.

Capillary separations, although delivering much improved sensitivity, especially when combined with mass spectrometry, often have the drawback of reduced robustness. This is partially due to the limited stability of packed capillary columns and the risk of clogging (same aspect as discussed above). Monolithic capillary columns made of polymeric (Svec et al., 2003) or silica-based materials promise to overcome some of the limitations mentioned above, namely that of packing stability. An interesting study was performed by the Guryca et. al. to provide a side-by-side comparison of monolithic nano-LC columns used in reversed-phase chromatography of proteins tryptic digests (Guryča et al., 2008). They compared PepMap (LC Packings, Amsterdam, The Netherlands, 3 μm 100Å, ID 75 μm, 15 cm), Chromolith CapRod (Merck KGaA, Darmstadt, Germany; silica monolith-C18, ID 100 μm, 15 cm) and PS-DVB (LC Packings; polystyrene monolith, ID 100 μm, 5 cm) columns (all C18 modification), in terms of the number of peptides identified and also with respect to their chromatographic characteristics. In terms of performance the peak shapes obtained on Chromolith CapRod and PepMap columns appeared to be very similar, and the peak widths for both columns were in the range 0.3–0.4 min. The PS-DVB column exhibited somewhat disappointing performance which could be attributed, to the mobile phase composition used. However, it was concluded, that generally the performance of both silica based columns was superior to that of monolithic PS-DVB (Guryča et al., 2008). Also a similar finding was observed comparing peptide identification power. Comparing column throughput Chromolith CapRod column was superior with 5.0 μl/min in contrast to flow rates of up to 0.8 μl/min for PS-DVB column and to 0.5 μl/min for the particulate (PepMap) column. Moreover, it was found that, for short gradients, the number of identifications is not affected by the flow rate (3–10 mm/s). The results shown demonstrate the greater potential of monolithic compared to particle-based columns, as higher flows can be utilized, enabling the number of identifications per unit of time to be significantly increased. Furthermore, due to their higher porosity they have fewer tendencies to get clogged. Usually, micro columns for LC are fabricated by packing beads with a controlled range of diameters and pore sizes. To obtain a better efficiency, columns have been packed with particles of ever smaller diameters (Szabolcs et al., 2009) bringing about another practical limitation: the increase of the back pressure. To circumvent this problem, alternative chromatographic modalities such as ultrahigh-pressure liquid chromatography, open tubular chromatography, and capillary electro-chromatography have been investigated. All this has led to the use of particle sizes in the range of 3 to 5 *µ*m as a good compromise between column efficiency and pressure drop. Moreover, it was demonstrated that the recently developed monolithic-type HPLC columns could be operated at high flow rates while maintaining a high efficiency. In this context,

and a volume of the bio-fluid injected. After injecting plasma column performance dropped drastically when the volume of half column volume was injected. For urine the column stability was at least 20 times higher. This is definitely related to the sample complexity. Comparing the life time of the same dimension of monolithic silica columns and particle packed columns under same conditions; monolithic silica column life time was at least double compared with particulate packed column of the similar dimension. This is not a surprise, as any particle packed column contains particles and frits to maintain particles in the column. The flow through between particles is much smaller that the particle size itself, for example, if the column packed with 2 µm particles the space between particles is about 0.5 µm. Monolithic silica column made as one single spongy rod, does not contains frits, and

Capillary separations, although delivering much improved sensitivity, especially when combined with mass spectrometry, often have the drawback of reduced robustness. This is partially due to the limited stability of packed capillary columns and the risk of clogging (same aspect as discussed above). Monolithic capillary columns made of polymeric (Svec et al., 2003) or silica-based materials promise to overcome some of the limitations mentioned above, namely that of packing stability. An interesting study was performed by the Guryca et. al. to provide a side-by-side comparison of monolithic nano-LC columns used in reversed-phase chromatography of proteins tryptic digests (Guryča et al., 2008). They compared PepMap (LC Packings, Amsterdam, The Netherlands, 3 μm 100Å, ID 75 μm, 15 cm), Chromolith CapRod (Merck KGaA, Darmstadt, Germany; silica monolith-C18, ID 100 μm, 15 cm) and PS-DVB (LC Packings; polystyrene monolith, ID 100 μm, 5 cm) columns (all C18 modification), in terms of the number of peptides identified and also with respect to their chromatographic characteristics. In terms of performance the peak shapes obtained on Chromolith CapRod and PepMap columns appeared to be very similar, and the peak widths for both columns were in the range 0.3–0.4 min. The PS-DVB column exhibited somewhat disappointing performance which could be attributed, to the mobile phase composition used. However, it was concluded, that generally the performance of both silica based columns was superior to that of monolithic PS-DVB (Guryča et al., 2008). Also a similar finding was observed comparing peptide identification power. Comparing column throughput Chromolith CapRod column was superior with 5.0 μl/min in contrast to flow rates of up to 0.8 μl/min for PS-DVB column and to 0.5 μl/min for the particulate (PepMap) column. Moreover, it was found that, for short gradients, the number of identifications is not affected by the flow rate (3–10 mm/s). The results shown demonstrate the greater potential of monolithic compared to particle-based columns, as higher flows can be utilized, enabling the number of identifications per unit of time to be significantly increased. Furthermore, due to their higher porosity they have fewer tendencies to get clogged. Usually, micro columns for LC are fabricated by packing beads with a controlled range of diameters and pore sizes. To obtain a better efficiency, columns have been packed with particles of ever smaller diameters (Szabolcs et al., 2009) bringing about another practical limitation: the increase of the back pressure. To circumvent this problem, alternative chromatographic modalities such as ultrahigh-pressure liquid chromatography, open tubular chromatography, and capillary electro-chromatography have been investigated. All this has led to the use of particle sizes in the range of 3 to 5 *µ*m as a good compromise between column efficiency and pressure drop. Moreover, it was demonstrated that the recently developed monolithic-type HPLC columns could be operated at high flow rates while maintaining a high efficiency. In this context,

flow through pores (macropores) are about 2 µm diameter.

(Kele & Guiochon, 2002) investigated the reproducibility of the preparation of the first columns getting reproducibilities higher than with particle based columns. Because of their capacity to perform fast separations they can be used for fast screening methods and applications in multidimensional chromatography systems. Conditioning and regeneration of these monolithic columns can be done in a short time when compared with the corresponding capillary packed columns, thus making more effective use of costly LC-MS equipment. They can be easily integrated in fully automated systems to perform unattended runs. These columns are flexible and they show a good performance at both low (1.5 µl/min) and high (4.5 µl/min) flow rates. Such flow rate range is highly compatible with MALDI plate spotting strategy. Fraction could be spotted directly, then the flow of 4 µl/min allows to spot up to 8 fractions. If the flow rate is set to 3 μl/min, and an equal flow of MALDI matrix solution is added post-column (7 mg/ml re-crystallized αcyanohydroxycinnamic acid, 2 mg/ml ammonium phosphate, 0.1% trifluoroacetic acid, 80% acetonitrile) and the combined eluant is automatically spotted onto a stainless steel MALDI target plate every 6 s (0.6 μl/spot), a total of 370 spots obtained per original SCX fraction (Fort et al., 2009). Haffey demonstrated similar approach and obtained 3828 MALDI-TOF spots from the 12 SCX fractions (Haffey et al., 2009). Such a separation strategy offers enormous discrimination power and imposing peak capacity.
