Stationary phases silica-based

Probably the best compromise for silica based stationary phases is to use corrected retention volume data for solutes eluted at a (k ) of greater than 5 and only compare chromatographic data for solutes of approximately the same molecular size. 

Silica stationary phases display some ion exchange properties, which may also influence the separation characteristics of silica. One of the main disadvantages of the use of silica and silica-based stationary phases is their instability even at slightly alkaline pH, such as 8.0. HPLC stationary phases can be characterized with the average particle diameter and the distribution of particle size. Smaller average diameter and narrow particle size distribution generally enhances the efficacy of separation. The average particle diameter can be calculated with different methods ... 

Silica-based stationary phases with a chemically bonded ligand on the surface can be characterized by the carbon content (grams of carbon per 100 g of packing) and by the bonding density (micromols of ligand bonded/square meter of initial silica surface area). 

Only the silica-based stationary phases with covalently bonded alkyl chain, cyano and propylamino ligands have found practical applications in HPLC. Besides these common ligands, the experimental use of naphthalene, pyrene and nitroaromatic as ligands has also been reported. Silica-based stationary phases with covalently bonded cyclodextrins or cyclodextrin derivatives have been frequently employed in the separation and quantitative determination of isomer pairs. 

Because of its higher pH stability, alumina and alumina-based stationary phases offer an alternative to silica and silica-based stationary phases. 

The separation of basic and metal-sensitive compounds is difficult on silica-based stationary phase materials, but these separations can be performed on vinyl alcohol copolymer gels. Examples are the separation of methallothionein from dolphin kidney, a-, j8-, and y-endorphin, and nucleotide and nucleoside mixtures.8 However, an analytical-scale separation may also be performed on surface-modified wide-pore silica gels (pore size 300 A or more), using columns which showed a negative response in the heavy metal test described above. 

B. Properties of Modern Silica-based Stationary Phases... 

One disadvantage of all silica-based stationary phases is their instability against hydrolysis. At neutral pH and room temperature the saturation concentration of silicate in water amounts to lOOppm. Solubility increases with surface area, decreasing particle diameter drastically with pH above 7.5. This leads also to a reduction of the carbon content. Hydrolysis can be recognized during the use of columns by a loss in efficiency and/or loss of retention. Bulky silanes [32], polymer coating [33], or polymeric encapsulation [34] have been used in the preparation of bonded phases to reduce hydrolytic instability, but most of the RPs in use are prepared in the classical way, by surface silanization. Figure 2.3 schematically shows these different types of stationary phases. 

Unfortunately, even this modified equation does not describe the true practical situation in LC, as it is complicated by the fact that all silica-based materials exhibit exclusion properties. The pore diameter of silica-based stationary phases can range from, perhaps, 2-3 Angstrom to as much as 1000-2000 Angstrom. Consequently, some, otherwise open pores, are accessible to the solute while others are not, depending on the size of the molecule. Therefore, only those pores that have a diameter equal to, or greater than, that of the solute molecules are accessible and only the stationary phase within those pores can effect retention. In addition, the static interstitial volume between the particles can also exhibit exclusion properties and some of the static interstitial volume may also be inaccessible to the larger solutes. As a consequence, equation (12) must be further modified to give,... 

It follows that retention measurements on silica based stationary phases for the purpose of obtaining thermodynamic data is fraught with difficulties. Data from solutes of different molecular size cannot be compared or related to other Interacting variables ideally, thermodynamic measurements should be made on columns that contain stationary phases that exhibit no exclusion properties. However, the only column system that might meet this requirement is the capillary column which, unfortunately introduces other complications wmcn will be discussed later. 

Although MCC is a chromatographically based technique, the process is generally considered to be environmentally friendly, as high production rates are possible, solvent loss is minimal (it is recovered during product isolation and recycled back into the system), and the silica-based stationary phase is reported to last at least 3-4 years. 

For high-pH operation of any silica-based stationary phase, temperature should not exceed 40°C, organic buffers should be used instead of phosphate or carbonate, and methanol instead of acetonitrile should be the organic solvent. 

Ion suppression is not often applied to strong acids or strong bases because of the extremes in pH that would be required for retention. Suppression by the addition of buffers is restricted to the range pH 3-8, because silica-based stationary phases are unstable in solutions of either high or low pH (i.e., above pH 8 or below pH 2). These restrictions do not apply to polymeric supports, and polymer-based stationary phases can be used in the separation of a wider range of solutes using the ion-suppression technique. 

Silica-base stationary phases have also been employed for enantiomeric separations in CEC [6,72-81]. In the initial work on chiral CEC, commercially available HPLC materials were utilized, including cyclodextrins [6,74,81] and protein-type selectors [73,75,80] such as human serum albumin [75] and ai-acid glycoprotein [73]. Fig. 4.9, for example, depicts the structure of a cyclodextrin-base stationary phase used in CEC and the separation of mephobarbital enantiomers by capillary LC and CEC in a capillary column packed with such a phase. The column operated in the CEC mode affords higher separation efficiency than in the capillary LC mode. Other enantiomeric selectors are also use in CEC, including the silica-linked or silica-coated macrocyclic antibiotics vancomycin [82,83] and teicoplanin [84], cyclodextrin-base polymer coated silicas [72,78], and weak anion-exchage type chiral phases [85]. Relatively high separation efficiency and excellent resolution for a variety of compounds have also been achieved using columns packed with naproxen-derived and Whelk-0 chiral stationary phases linked to 3 pm silica particles [79]. Fig. 4.10 shows the... 

If a buffer is present in the mobile phase, then the dissociation will be controlled by the pKa value of the buffer. Because of the pH limitations of silica-based stationary phases, only weak acids can be used as buffer compounds. As an example, we will consider the dissociation of a weak acid (HA) in the presence of a buffer acid (HB). 

The growing popularity of reversed phase chromatography in particular has prompted polymer manufacturers to investigate the use of polymeric media for this mode of operation. Macroporous copolymers of styrene and divinylbenzene have similar properties to silica based stationary phases bonded with alkyl chains. However, the absence of leachables and stability at high pH can offer advantages under certain circumstances. High quality, mechanically stable macroporous polymeries are now manufactured at much larger scales than the... 

As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds. Peptides are also readily purified by gradient elution on normal phase silica, moving from acetonitrile to aqueous mobile phase 3,2l This technique is particularly useful for extremely hydrophilic peptides that are not strongly retained on reversed phase media. 

Silica based stationary phases are unstable to alkaline conditions due to loss of the alkyl chain and dissolution of the silica... 

---