Chemically bonded phases, liquid chromatograph

Berendsen, GE. and de Galan, L., Preparation and chromatographic properties of some chemically bonded phases for reversed-phase liquid chromatography, J. Liq. Chromatogr, 1, 561, 1978. 

The influence of the bonded organic moiety on solute retention has not yet been elucidated and only a very small number of papers discuss the properties and use of such phases so far. The numerous advantages of chemically bonded phases make the application of polar chemically bonded phases with nonpolar eluents quite attractive even if the standardization of these phases may pose problems 106) similar to those encountered in the standardization of aidsorbents as well as of polymeric liquid phases in gas chromatography. A detailed discussion of the properties and chromatographic use of bonded stationary phases is given by Melander and Horvath (this volume). 

Chemically bonded phases (CBP s) are very commonly used in LC, and occasionally also in GC. Such phases cannot be seen as either a solid or a liquid. The common term [201] used for LC involving such phases is bonded phase chromatography (BPC). To be consistent, the stationary phase identification should follow that of the mobile phase in defining the chromatographic system. Hence, LBPC should be used for liquid chromatography using chemically bonded stationary phases. 

The major chromatographic techniques have been included. However, the book does not intend to give a comprehensive overview of the historic developments in separation science, and some classical techniques that are not in use today have not been covered. An example is paper chromatography, which was replaced by the more efficient thin layer chromatography a long time ago. Another example is column liquid-liquid partition chromatography, which more or less disappeared after the introduction of chemically bonded phases in HPLC. 

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. 

Wise SA, Chesisr SN, Hertz HS, Hilpert LR, and May WE (1977) A chemically bonded amino-silane stationary phase for the high-performance liquid chromatographic separation of polynuclear aromatic compounds. Anal Chem 49 2306-2310. 

Yang, W.-H., Chen, I-L., and Wu, D.-H., Chemically bonded phenylsilicone stationary phases for the liquid chromatographic separation of polycyclic aromatic hydrocarbons and cyclosiloxanes, /. Chromatogr. A, 722, 97, 1996. 

HPLC techniques were initially developed as liquid-liquid chromatographic methods and difficulties in maintaining the stationary phase were resolved by chemically bonding it to the particulate support. Subsequently a whole range of column materials have been developed that enable the basic HPLC instrumentation to be used for the major chromatographic techniques. 

Precoated plates are also available for reversed-phase liquid-liquid partition thin-layer chromatography. Here the silica gel has been treated with an octadecyl silylating reagent thus coating the particles with a non-polar chemically-bonded thin film. The solvent employed is more polar than the film and chromatographic development results from partition between these two phases. 

A liquid chromatographic method is utilized for the determination of clopidogrel bisulfate in samples of the bulk drug substance. The method uses a column (L57 column size 15 cm x 4.6 mm) packed with ovomucoid (a chiral-recognition protein) that is chemically bonded to silica particles of 5 /im diameter and a pore size of 120 A. Both the reference standard and the sample to be analyzed are dissolved in methanol, and then diluted with mobile phase. The mobile phase is 75 25 0.01 M phosphate buffer /acetonitrile, and the flow rate is adjusted to 1.0 ml/min. Observation is made on the basis of the UV absorbance at 220 nm, and the clopidogrel peak has a relative retention time about 1.0 min. 

S. N. El-Gizawy, A. N. Ahmed, and N. E. El-Rabbat, High performance liquid chromatographic determination of multivitamin preparations using a chemically bonded cyclodextrin stationary phase, Anal. Lett., 24 1173 (1991). 

Figure 1. Liquid chromatographic sefxiration of an ethyl acetate liver extract of barbiturates. Column reverse phase, n-octadectjl groups chemically bonded to 10 p. silica mobile phase methanol/water.

Procedure (See Chromatography, Appendix IIA.) Use a high-performance liquid chromatograph equipped with an ultraviolet detector that measures absorption at 254 nm and a 25- to 30-cm x 4-mm (id) stainless-steel column, or equivalent, packed with octadecyl silane chemically bonded to porous silica or ceramic microparticles 5 to 10 pun in diameter, or equivalent. Maintain the mobile phase at a pressure and flow rate capable of giving the required resolution (see below). Inject a volume, up to 25 pL, of the System Suitability Solution in a similar manner. Calculate the resolution, R (>3.6), between calcium formyltetrahydrofolate and Folic Acid by the equation... 

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