Monolithic columns retentivity

We use the second-dimension separation from Fig. 6.6 with a 25 pL injection volume and 2.5 min sampling time the separation is an RPLC method that uses a monolithic column. Thus, 10 pL/min is the maximum flow rate in the first-dimension. Fig. 6.7 shows the development of the first-dimension column that utilizes a hydrophilic interaction (or HILIC) column for the separation of proteins at decreasing flow rates. The same proteins were separated in Fig. 6.6 (RPLC) and 6.7 (HILIC) and have a reversed elution order, which is known from the basics of HILIC (Alpert, 1990). It is believed that HILIC and RPLC separations are a good pair for 2DLC analysis of proteins as they appear to have dissimilar retention mechanisms, much like those of NPLC and RPLC it has been suggested that HILIC is similar in retention to NPLC (Alpert, 1990). Because the HILIC column used in Fig. 6.7 gave good resolution at 0.1 mL/min and no smaller diameter column was available, the flow was split 10-fold to match the second-dimension requirement... 

Kele, M., Guiochon, G. (2002). Repeatability and reproducibility of retention data and band profiles on six batches of monolithic columns. J. Chromatogr. A 960, 19 -49. 

Chirica and Remcho first created the outlet frit, packed the column with ODS beads, and then fabricated the inlet frit. The column was filled with aqueous solution of a silicate (Kasil) and the entrapment achieved by heating the column to 160 °C [105,106]. The monolithic column afforded considerably reduced retention times compared to the packed-only counterpart most likely due to a partial blocking of the pores with the silicate solution. This approach was recently extended to the immobilization of silica beads in a porous organic polymer matrix [107]. 

The secondary structure, the mesopores, is similar to the internal structure of standard HPLC particles. This secondary structure provides the surface for retention. The standard pore size is in the order of 13 nm, resulting in a specific surface area of about 300 mVg. Due to the lower ratio of retentive structure to interstitial space, the retentivity of monoliths and the preparative loadability tends to be significantly lower than the retentivity and loadability of packed beds of 10-nm particles. Since the monolithic columns described here are made from silica, they can be derivatized in the same way and with the same technology as silica-based particles. Also, the useful pH range is the same as for silica-based particles. 

Kele, M. and Guiochon, G. Repeatability and Reproducibility of Retention Data and Band Profiles on Six Batches of Monolithic Columns,/. Chromatogr., 960 19—49,2002. 

Current research in CEC involves the use of monolith capillaries, which are fritless, packed capillaries having stationary phase bound to the capillary wall. Using porous polymer monoliths, the retention of a packed column can be found in an open tubular capillary. In general, CEC remains unsettled. Frit technology is unreliable and research into monolithic capillaries is still a work in progress. Recent progress in CEC can be found in the reviews by Colon and co-workers. 

New concepts presented in this edition include monolithic columns, bonded stationary phases, micro-HPLC, two-dimensional comprehensive liquid chromatography, gradient elution mode, and capillary electromigration techniques. The book also discusses LC-MS interfaces, nonlinear chromatography, displacement chromatography of peptides and proteins, field-flow fractionation, retention models for ions, and polymer HPLC. 

Numerous studies have been made characterizing the performance of silica-based rod columns [69], demonstrating the high level of reproducibility of analytical retention data [68] and of isotherm data [70], showing that the thermodynamic properties of the interactions between various solutes and chemically bonded Cig silica were very similar whether the silica support was made of particulate or monolithic material [71]. It has also been shown that the mass transfer kinetics was very similar for particulate and monolithic columns [72-74]. Even the satu-... 

Almost all stationary phases used in chromatography have a bimodal pore size distribution. The first mode corresponds to the macropores or throughpores that allow the percolation of the column by the stream of mobile phase. The second distribution corresponds to the mesopores that combine to give the conventional internal porosity distribution described in the previous section. The mesopores are responsible for most of the specific surface area necessary to provide the retention and the saturation capacity that are needed to permit the retention of the mixture components in a good solvent, a condition for chromatographic separation. Nonporous particles have been used with only moderate success because very weak solvents must be used to achieve sufficient retention, which often causes solubility problems, and the saturation capacity of these particles is small. The terms of macro- and meso-pores apply as well to columns made of packed particles and to monolithic columns. 

EC differs from MEKC in that it uses a real stationary phase. In essence, it is conventional LC in which the mobile phase is driven by electro-osmotic flow rather than pressure. However, as in MEKC, the mechanism of separation is mixed, with electrophoretic mobility affecting the retention of charged solutes. The stationary phase particles can be very small, as there is no pressure drop in the column. Typically, 1.5 /rm particles of Cig-modified silica are used. Promising results were also obtained with monolithic columns for EC. Capillary EC provides about twice as many plates as HPLC for the same particle size and column length. 

In recent years, monolithic columns have been developed. While classical columns are packed with particles, monolithic columns are continuous structures that look like corrals. The skeleton can be porous, like particles, and contains the surface area responsible for retention. The space between the skeleton provides the flow passages. The key advantage of monolithic columns lies in the fact that the interstitial space can be manipulated independently of the domain size that is responsible for retention. Therefore, the compromise between column... 

These two disadvantages are less pronounced with solid core particles than with silica-based monolithic phases. Moreover, the retention behavior of every monolithic column can be considered one of a kind given the manufacturing process for the bonding. There are three more considerations with monolithic columns ... 

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