Bonded stationary phases

Grushka G (ed) (1974) Bonded Stationary Phases in Chromatography. Ann Arbor Science, Ann Arbor, Mich. 

With the development of HPLC, a new dimension was added to the tools available for the study of natural products. HPLC is ideally suited to the analysis of non-volatile, sensitive compounds frequently found in biological systems. Unlike other available separation techniques such as TLC and electrophoresis, HPLC methods provide both qualitative and quantitative data and can be easily automated. The basis for the HPLC method for the PSP toxins was established in the late 1970 s when Buckley et al. (2) reported the post-column derivatization of the PSP toxins based on an alkaline oxidation reaction described by Bates and Rapoport (3). Based on this foundation, a series of investigations were conducted to develop a rapid, efficient HPLC method to detect the multiple toxins involved in PSP. Originally, a variety of silica-based, bonded stationary phases were utilized with a low-pressure post-column reaction system (PCRS) (4,5), Later, with improvements in toxin separation mechanisms and the utilization of a high efficiency PCRS, a... 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

The most common technique used for agrochemicals is reversed-phase SPE. Here, the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon (e.g. C4-C18) or styrene-divinylbenzene copolymer. This technique operates in the reverse of normal-phase chromatography since the mobile phase is polar in nature (e.g., water or aqueous buffers serve as one of the solvents), while the stationary phase has nonpolar properties. 

Liquid-solid chromatography (LSC), sometimes referred to as normal phase or straight phase chromatography, is characterized by the use of an inorganic adsorbent or chemically bonded stationary phase with polar functional groups and a nonaqueous mobile phase... 

Basiuk, V. A. and Chuko, A. A., Selectivity of bonded stationary phases containing uracil derivatives for liquid chromatography of nucleic acid components, /. Chromatogr. Sci., 31, 120, 1993. 

In NPLC, which refers to the use of adsorption, i.e. liquid-solid chromatography (LSC), the surface of microparticulate silica (or other adsorbent) constitutes the most commonly used polar stationary phase normal bonded-phase chromatography (N-BPC) is typified by nitrile- or amino-bonded stationary phases. Silica columns with a broad range of properties are commercially available (with standard particle sizes of 3, 5 and 10 im, and pore sizes of about 6-15nm). A typical HPLC column is packed with a stationary phase of a pore size of 10 nm and contains a surface area of between 100 and 150m2 mL-1 of mobile phase volume. 

Surfactants are separated according to adsorption or partitioning differences with a polar stationary phase in NPLC. This retention of the polar surfactant moiety allows for the separation of the ethylene oxide distribution. Of all the NPLC packings that have been utilized to separate nonionic surfactants, the aminopropyl-bonded stationary phases have been shown to give the best resolution (Jandera et al., 1990). The separation of the octylphenol ethoxylate oligomers on an amino silica column is shown in Fig. 18.4. Similar to the capabilities of CE for ionic surfactants, the ethylene oxide distribution can be quantitatively determined by NPLC if identity and response factors for each oligomer are known. 

Locke, D. (1974) Selectivity in reversed-phase liquid chromatography using chemically bonded stationary phases. J. Chromatogr. Sci. 12, 433 137. 

FEURLE, J., JOMAA, H., WILHELM, M GUTSCHE, W.B., HERDRICH, M., Analysis of phosphorylated carbohydrates by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry utilising p-cyclodextrin bonded stationary phase, J. Chromatogr., 1998,803,111-119. 

Variations in retention and selectivity have been studied in cyano, phenyl, and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline, and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile, or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns were correlated with polar group selectivities of mobile-phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers [84],... 

Perfluorinated stationary phases offer superior selectivity in comparison to the current hydrocarbon bonded-stationary phases. 

Packings for HPLC can be further described as either pellicular or porous. Pellicular particles are made from spherical glass beads, which are then coated with a thin layer of stationary phase. For example, a porous layer can be deposited onto the glass bead to produce a porous layer or a superficially porous particle. The porous layer can in turn be coated with liquid stationary phase or reacted to give a bonded stationary phase. Pellicular particles are generally less efficient than the porous layer of superficially porous particles. 

The most popular and versatile bonded phase is octadecylsilane (ODS), n-C18H37, a grouping that is non-polar and used for reverse phase separations. Octylsilane, with its shorter chain length, permits faster diffusion of solutes and this results in improved peak symmetry. Other groups are attached to provide polar phases and hence perform normal phase separations. These include cyano, ether, amine and diol groups, which offer a wide range of polarities. When bonded stationary phases are used, the clear distinction between adsorption and partition chromatography is lost and the principles of separation are far more complex. 

The great versatility of HPLC lies in the fact that the stability of the chemically bonded stationary phases used in partition chromatography allows the use of a wide range of liquids as a mobile phase without the stationary phase being lost or destroyed. This means that there is less need for a large number of different stationary phases as is the case in gas chromatography. The mobile phase must be available in a pure form and usually requires degassing before use. The choice of mobile phase (Table 3.6) is influenced by several factors. 

Separation and quantitation of carbohydrate mixtures may be achieved using HPLC, a method that does not necessitate the formation of a volatile derivative as in GLC. Both partition and ion-exchange techniques have been used with either ultraviolet or refractive index detectors. Partition chromatography is usually performed in the reverse phase mode using a chemically bonded stationary phase and acetonitrile (80 20) in 0.1 mol U1 acetic acid as the mobile phase. Anion- and cation-exchange resins have both been used. Carbohydrates... 

Figure 3.23 Selectivity of phenyl and alkyl bonded stationary phase materials for protein separation. Column A, TSK gel phenyl-5PW RP, 75 mm x 4.6 mm i.d. B, TSK gel TMS 250, 75 mm x 4.6 mm i.d. eluent, 60 min linear gradient elution from 5% of 0.05% trifluoroacetic acid in 5%> aqueous acetonitrile to 80% of 0.05% trifluoroacetic acid in 80% aqueous acetonitrile flow rate, lml min-1 detection, UV 220 nm. Peaks 1, ribonuclease 2, insulin-, 3, cytochrome c 4, lysozyme-, 5, transferrin-, 6, bovine serum albumin-, 1, myoglobin-, and 8, ovalbumin.

Torok, G. et al.. Direct chiral separation of unnatural amino acids by high performance liquid chromatography on a ristocetin A-bonded stationary phase. Chirality, 13, 648, 2001. 

Jinno, K., Effect of the aUcyl chain length of the bonded stationary-phase on solute retention in reversed phase high performance hquid chromatography, Chromato-graphia, 15, 667, 1982. 

---