Mixed-Mode Bonded Phases

Acclaim HILIC-10 is similar to Acclaim Mixed-Mode WAX-1. This relatively new column is also based on high-purity spherical silica covalently modified with a proprietary hydrophilic layer. It is suited for use in a broad range of applications including the separation of hydrophilic drugs and their metabolites. Acclaim Mixed-Mode HILIC-1 is a stationary phase that combines both RP and HILIC properties. Unlike traditional RP or HILIC columns, this packing material features a long alkyl chain with a diol functionality at the terminal end. 

Because traditional HILIC columns such as underivatized silica, cyano-, amino-, and diol-bonded phases have hydrophilic surfaces, they are incapable of separating small molecules via hydrophobic interaction. By comparison. 

Brand name Manufacturer Particle size (pm) Pore size (A) Surface area (m /g) 

Acclaim Mixed-Mode Thermo Fisher Scientific 3,5 120 300 

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A pivotal step in the analytical process is sample preparation. Frequently liquid-liquid extractions (LLEs) are used. Solvents, pH, and multiple back extractions are all manipulated to increase selectivity and decrease unwanted contaminants before injection on the GC system. Solid phase extraction (SPE) is more convenient than it used to be because of an increase in commercially available SPE columns. SPE columns are packed with an inert material that binds the drug of interest, allowing impurities to pass through. As with LEE, solvent choices and pH affect retention and recovery. There are three commercially available types of SPE columns, diatomaceous earth (which uses the same principles as LLE), polystyrene-divinylbenzene copolymer, and mixed mode bonded silica (Franke and de Zeeuw, 1998). 

The CEC phases must be capable of carrying a charge to generate an EOE and appropriate moieties to facilitate the chromatographic processes. Silica-based reversed-phase packing materials have been most widely used in CEC. The use of polymeric and mixed-mode bonded particles has also been reported. Eor the silica-based phases, the carbon chains bonded on the silica surface provide the retention and selectivity for analytes, and the residual silanol groups on the surface of the silica are ionizable and generate the EOF. 

Mills, M.S., E.M. Thurman, and M.J. Pedersen (1993). Application of mixed-mode, sohd-phase extraction in environmental and clinical chemistry. Combining hydrogen-bonding, cation-exchange and Van der Waals interactions. J. Chromatogr., 629 11-21. 

Fig. 4.12. EOF velocity as a function of the mobile phase pH for a C18 bonded silica and a mixed-mode bonded silica (SCX/C18). Reprinted from ref. [101] with permission. Copyright Elsevier 2000.

Mills, M. S. Thurman, E. M. and Pedersen, M. J. 1993. Mixed-mode solid-phase extraction Combining mechanisms of interaction hydrogen bonding, cation exchange, and reverse phase, J. Chromatog., 629 11-21. 

One of the key aspects of using the multiligand approach for synthesizing mixed-mode stationary phases has been the reproducibility of the bonding chemistry. In most phases produced a kinetic approach has been taken. Two or more ligands of the appropriate starting silane are simultaneously co-... 

Besides the above differentiation, restricted-access media can be further subdivided on the basis of the topochemistry of the bonded phase. Packings with a uniform surface topochemistry show a homogenous ligand coverage, whereas packings with a dual topochemistry show a different chemical modification of the pore internal surface and the particle external surface (114). Restricted-access media of the former type are divided into mixed-mode and mixed-function phases, bonded-micellar phases, biomatrix, binary-layered phases, shielded hydrophobic phases, and polymer-coated mixed-function phases. Restricted-access media of the latter type include the Pinkerton s internal surface reversed-phase, Haginaka s internal surface reversed-phase diol, alkyl-diol silica, Kimata s restricted-access media, dual-zone phase, tris-modified Styrosorb, Svec s restricted-access media, diphil sorbents, Ultrabiosep phases. Bio Trap phases, and semipermeable surface phases. 

As we mentioned above, even end-capped packings have some free silanols, either left over from incomplete binding or by hydrolysis of the bonded phase. These give a reverse-phase separation a mixed mode nature. Most of the separation is due to the nonpolar partitioning bonded phase, but some of it comes from these ionizable, polar silanols. Metal cations form a pair bond couple and lock the silanol into the ionized form the partition separation changes. 

Bond Elut (Agilent) and CBA ISOLUTE (International Sorbent technology) both representing an irregular end-capped mixed-mode phase of a weak cation exchanger (carboxylic acid) and a mid-polarity sorbent. /V-butyl-scopolamine was extracted accordingly from equine urine [78], Elution of analytes was achieved by the use of a methanolic solution acidified either with 1 M HC1 [54] or 1 % formic acid [78] allowing competition between QTA and hydronium ions. Recoveries were between 96 and 103 % [54] (Table 4). 

Fig. 4.11. (A) Generic representation of silane chemistry used to bond stationary phases onto silica supports. (B) Examples of packing materials that will enhance EOF. (C) Mixed-mode phase designed by El Rassi s group. Adapted from ref. [95] with permission. Copyright Wiley-VCH 1998.

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