Silica-Based Mixed-Mode Columns

Technologies, are zwitterionic type mixed-mode columns with either RP or normal-phase (NP) characteristics. Obelise R incorporates a cationic group close to the silica surfece separated from an anionic group by a hydrophobic chain, while in Obelise N the two functional groups are separated by a hydrophilic chain. 

Zwitterionic mbeed-mode columns are also offered by Merck Millipore (Darmstadt, Germany) under the trade name SeQuant ZIC-HILIC. Separations of polar and ionizable compounds on this sulfobetain-type bonded phase that is available in a 3 and 5 pm format, are achieved by hydrophilic partitioning combined with weak ionic interactions. 

Type IV materials exhibit unique ion-exchange properties supplemented by RP characteristics, so that they may be considered as RP-modified ion-exchange materials. Lammerhofer et al. [59] have developed a number of RP/weak anion exchanger (WAX) materials consisting of a selector immobilized onto thiol-modified silica. In these phases, the WAX site is located at the top of the lipophilic layer and is linked to the silica support via a lipophihe spacer with polar-embedded amide or sulfide groups. These columns, however, are not commercially available. 

More than 5 years ago, two type IV mixed-mode columns that are based on an alkyl chain with terminal ion-exchange functional groups were commercialized under the trade name Acclaim Mixed-Mode WAX-1 [67] and Acclaim 

Mixed-Mode WCX-1 [68] (Thermo Fisher Scientific, Waltham, MA, USA). The Acclaim Mixed-Mode WAX-1 column combines both hydrophobic and anion-exchange characteristics, which facilitates selectivity adjustment by changing mobile-phase ionic strength, pH, or organic modifier, either independently or concurrently. 

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All these examples demonstrate the superior separation capabilities of this silica-based mixed-mode column with its novel column chemistry and unique selectivity. 

Silica-based mixed mode column packing materials are not yet commercially available. At present it seems as if mixed mode chromatography is useful only for very specialized problems, for example separation of sequence isomeric oligonucleotides. 

In fact, every silica-based reversed phase represents a mixed-mode column, because besides the alkyl chains it has silanol groups, which are groups capable of weak cation exchange. Some C18 materials of the first generation, such as Partisil ODS from Whatman or Spherisorb ODSl from Waters, have only a very low carbon content (<5%) and a high number of free silanol groups. With these... 

Nogueira, R., Lubda, D., Leitner, A., Bicker, W, Maier, N.M., Lammerhofer, M., and Lindner, W, Silica-based monohthic columns with mixed-mode reversed-phase/weak anion-exchange selectivity principle for high-performance liquid chromatography, J. Sep. ScL, 29, 966, 2006. 

Selectivity Control by Mobile Phase pH and Column Temperature These two parameters are of major importance for selectivity control in HPLC, especially with acidic and/or basic analytes. The charge state of analyte molecules influences both their hydrophobic (dispersion type) interaction, as well as possible ionic secondary interactions (e.g., with dissociated residual silanol group on silica-based stationary phases). The relatively wide range of so-called mixed-mode phases make very effective use of ionic interactions, but specialty phases are outside the scope of this chapter (refer to Chapter 4 for more information). 

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. 

Figure 10.201 Gradient elution of anionic surfactants in a shampoo using mixed-mode liquid chromatography on a silica-based column. Separator column Acclaim Surfactant Plus,...

The cationic melamine can be separated on Acclaim Mixed-Mode WCX-1 (see Section 6.7.2) using an ammonium acetate buffer as the eluent. This column features a mixed-mode silica-based packing material that incorporates... 

Sample introduction is a major hardware problem for SFC. The sample solvent composition and the injection pressure and temperature can all affect sample introduction. The high solute diffusion and lower viscosity which favor supercritical fluids over liquid mobile phases can cause problems in injection. Back-diffusion can occur, causing broad solvent peaks and poor solute peak shape. There can also be a complex phase behavior as well as a solubility phenomenon taking place due to the fact that one may have combinations of supercritical fluid (neat or mixed with sample solvent), a subcritical liquified gas, sample solvents, and solute present simultaneously in the injector and column head [2]. All of these can contribute individually to reproducibility problems in SFC. Both dynamic and timed split modes are used for sample introduction in capillary SFC. Dynamic split injectors have a microvalve and splitter assembly. The amount of injection is based on the size of a fused silica restrictor. In the timed split mode, the SFC column is directly connected to the injection valve. Highspeed pneumatics and electronics are used along with a standard injection valve and actuator. Rapid actuation of the valve from the load to the inject position and back occurs in milliseconds. In this mode, one can program the time of injection on a computer and thus control the amount of injection. In packed-column SFC, an injector similar to HPLC is used and whole loop is injected on the column. The valve is switched either manually or automatically through a remote injector port. The injection is done under pressure. 

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