Silica-based stationary phases monolithic columns

A typical mobile-phase composition is an acetonitrile-water gradient with a fixed concentration of trifluoroacetic acid (TFA), formic, or acetic acid (typically 0.05-0.5%). TFA acts as an ion-pairing agent and masks secondary interactions with the silica-based stationary phase. TFA may significantly suppress the ESI response in positive-ion mode. To avoid this, either formic acid is preferred or a mixture of 0.02% TFA and 0.5% acetic acid can be used. Some silica-based RPLC materials can be used with a lower TFA concentration (PepMap ). Alternatively, poly(styrene-divinylbenzene) polymeric materials (PS-DVB) can be applied. With a monolithic PS-DVB column, only a small decrease in separation efficiency on the monolithic column was observed when the TFA concentration was reduced from 0.2%to0.05%[51]. 

The use of monolithic columns in LC has advanced rapidly since their first introduction in the 1990s [18-21]. In contrast to capillary columns packed with particulate stationary phases, monolithic columns consist of a single continuous support. Monolithic stationary phases can be subdivided in two classes, i.e., polymer-and silica-based materials. 

A monolithic silica-based CIS stationary phase was used under high flow rate condition (2 mL/min) without significant back pressure in IPC analysis of a recently discovered new drug candidate for the treatment of Alzheimer s disease [15]. Nanoscale IPC using a monolithic poly(styrene-divinylbenzene) (PS-DVB) nanocolumn coupled to nanoelectrospray ionization mass spectrometry (nano-ESl-MS) was evaluated to separate and identify isomeric oligonucleotide adducts. Triethylammonium bicarbonate was used as the IPR. Interestingly, the performance of the polymeric monolithic PS-DVB stationary phase significantly surpassed that of columns packed with the microparticulate sorbents CIS or PS-DVB [16]. 

Improvements in the selectivity of the separation of microcystins and nodnlarin have been achieved by selecting the most efficient stationary phase, with this aim (Spoof 2002) compared a monolithic C-bonded silica rod colnmn (Merck Chromolith) to particle-based C and antide C 18 18 16 sorbents in the HPLC separation of eight microcystins and nodularin-R. Two gradient mobile phases of aqneons trillnoroacetic acid modified with acetonitrile or methanol, different flow-rates, and different gradient lengths were tested. The performance of the Chromolith colunrn measured the resolution of some microcystin pairs. The selectivity, efficiency (peak width), and peak asymmetry equalled, or exceeded, the performance of traditional particle-based columns. The Chromolith 21 colnmn allowed a shorteiting of the total analysis time to 4.3 minutes with a flow rate of 4 ml/minute. 

Another approach is the use of monolithic columns consisting of silica based rods of bimodal pore structure. They contain macropores (-1-2 pm) and smaller mesopores ( 10-20nm) [38]. The macropores allow for low backpressure at high flow rates. The mesopores provide the needed surface area for interactions between the solute and stationary phase. The macropores result in higher total porosity as compared to porous silica particles. Flow rates of 5 mL/min can be tolerated on a 10-cm column without an appreciable loss in... 

Instead of packed columns, monolithic (continuous bed), analytical, or capillary columns in the form of a rod with flow-through pores offer high porosity and improved permeability. Silica-based monolithic columns are generally prepared by gelation of a silica sol to a continuous sol-gel network, onto which a Cjg or another stationary phase is subsequently chemically bonded. Such columns provide comparable efficiency and sample capacity as conventional columns packed with 5-pm particle materials, but have three to five times lower flow resistance, thereby allowing higher flow rates and fast HPLC analyses. Rigid polyacrylamide, polyacrylate, polymethacrylate, or polystyrene monolithic columns are prepared by in sim polymerization. 

At present it seems that immobilization of silica-based particles within a packed capillary by hydrothermal treatment or sol-gel adhesion represent a simpler approach to the preparation of silica-based monoliths for capillary electrochromatography [302,332-334]. Particle fixation is achieved through adhesion by silica precipitated in the interparticle space released from the particles by hydrothermal treatment, or formed by hydrolysis and polycondensation of a solution of alkoxysilanes (sol-gel process). Since only relatively low temperatures are used in both processes, chemically bonded phases can be immobilized as easily as silica. The selectivity and separation efficiency of immobilized particle beds is generally similar to that of slurry packed columns prepared from the same stationary phases. 

The main advantages of monolithic columns are the superior separation performance and low flow resistance. In addition due to their continuous nature, frits are not required to retain the stationary phase. The production process of monolithic columns is more flexible than that of packed columns e.g., photo-polymerization can be applied to prepare monolithic structures or add selectivity locally. Both polymer- and silica-based monolithic capillary columns have been used for highly efficient separations in LC-mass spectrometry (MS) applications for proteomic research [24,25]. 

Monolithic silica is the most recently introduced class of stationary phases for electrochromatography on microchips. They benefit from their very high surface area, adjustable pore size, and controllable surface chemistry. Functionalities required for the separation in reversed-phase mode are typically incorporated onto the silica monolith by including an appropriate silicon alkoxide in the precursor mixture or by silanization of the surface after the monolith has been formed. Monolithic silica-based columns are generally known to exhibit superior performance in HPLC separations of small molecules, which is atftibuted to the presence of mesopores entailing large surface areas. 

As in GC, the uses of LC for the separation of chiral species have significantly increased. Column materials now include chiral phases that may, for example, be based on monolithic silica columns with chemically bonded beta-cyclodextrin, teicoplanin, or cellulose tris(3,5-dimethylphenylcarbamate). Hydro-phobic amino compounds have been separated by LC using a crown ether dynamically coated chiral stationary phase. 

In recent years, the interest in using porous silica and polymer-based monolithic stationary-phase media for ion chromatographic separations of inorganic and organic ions has increased.As compared to particle bed columns, monolithic columns represent a single piece of porous cross-linked polymer or porous silica. Monoliths are made in different formats as porous rods, generated in thin capillaries or made as thin membrane or disks. 

Reversed-phase chromatography is a separation method based on the hydrophobicity of the protein. In RPC, the hydrophobic stationary phase is based on silica gel or a synthetic polymer. In recent years, instead of bulk materials for column packing, polymer- or silica gel-based monolithic stationary phases have also been used [43]. 

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