Stationary phase HPLC

Nowadays, almost all commercially available HPLC stationary phases are also applicable to planar chromatography. In addition to the polar hydroxyl groups present on the surface of native silica, other polar functional groups attached to the silica skeleton can also enter into adsorptive interactions with suitable sample molecules (34). Silica with hydrophilic polar ligands, such as amino, cyano, and diol functions, attached to the silica skeleton by alkyl chains, all of which have been well proven in HPLC, have also been developed for TLC (34). 

Bolck, A. and Smilde, A. K., Multivariate characterization of RP-HPLC stationary phases, in Retention and Selectivity in Liquid Chromatography, Smith, R. M., Ed., Elsevier Science, Amsterdam, 1995, chap. 12. 

Few stationary phases optimised for pSFC (use of conventional HPLC stationary phases)... 

Szabo, G., Guczi, J., Bulman, R.A. (1995) Examination of silica-salicylic acid and silica-8-hydroxyquinoline HPLC stationary phases for estimation of the adsorption coefficient of soil for some aromatic hydrocarbons. Chemosphere 30, 1717-1727. 

Szabo, G., Guczi, J., Kodel, W., Zsolnay, A., Major, V., Keresztes, R (1999) Comparison of different HPLC stationary phases for determination of soil-water distribution coefficient, KqC, values of organic chemicals in RP-HPLC system. Chemosphere 39, 431 142. 

Size-exclusion HPLC (SE-HPLC) separates proteins on the basis of size and shape. As most soluble proteins are globular (i.e. roughly spherical in shape), separation is essentially achieved on the basis of molecular mass in most instances. Commonly used SE-HPLC stationary phases include silica-based supports and cross-linked agarose of defined pore size. Size-exclusion systems are most often used to analyse product for the presence of dimers or higher molecular mass aggregates of itself, as well as proteolysed product variants. 

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 ... 

Non-silica-based RP-HPLC stationary phases have also been developed and their separation capacity has been compared with those of silica-based ones. The porous structure of crosslinked polymer gels may be responsible for the markedly different selectivity and retention characteristics. Up till now, the mode of separation on polymer stationary phases is not entirely understood at the molecular level. It has been established that the size-exclusion effect may influence the retention of analyses on polymer gels. 

Besides silica, silica-based and polymeric stationary phases, porous graphitized carbon (PGC), zirconium oxide and its derivatives, alumina and its derivatives have been used for the solution of special separation problems which cannot be easily solved by using traditional HPLC stationary phases. 

Ansarian, H.R., Derakhshan, M., Rahman, M.M., Saknrai, T., Takafnji, M., Tanigu-chi, I., and Ihara, H., Evalnation of microstructuralfeatnres of a new polymeric organic stationary-phase grafted on silica surface a paradigm of characterization of HPLC-stationary phases by a combination of suspension-state H-1 NMR and solid-state C-13-CP/MAS-NMR, Anal. Chim. Acta, 547,179, 2005. 

Egelhaaf, H.J., OeUsrug, D., Pursch, M., and Albert, K., Combined fluorescence and NMR studies of reversed HPLC stationary phases with different ligand chain lengths, J. Fluorescence, 7, 311, 1997. 

The mobile phase controls HPLC separation While the HPLC stationary phase provides retention and influences the separation mechanism, it is the mobile phase which controls the overall separation. HPLC method development efforts focus on finding a set of mobile phase conditions that provide adequate separation of the analyte peak(s) from other components in the sample. 

The application of biosensors for process monitoring has been dogged by problems with fouling, selectivity, degrading bioactivity and long-term stability. Recent developments in molecularly imprinted polymers (MIPs) show some promise as synthetic receptors and have been used for this purpose for assays and as HPLC stationary phases. Recent work has shown that MIPs show increased robustness, storage endurance and lower cost compared with biosensors, which... 

Bassler BJ, Hartwick RA. 1989. The application of porous graphitic carbon as an HPLC stationary phase. J Chromatogr Sci 27(4) 162-165. 

Pagliara, A., Khamis, E., Trinh, A., Carrupt, P.A., Tsai, R.S. and Testa, B. (1995) Structural properties governing retention mechanisms on RP-HPLC stationary phase used for lipophilicity measurements. Journal of Liquid Chromatography, 18, 1721-1745. 

Even if MIP and BET are widely accepted regarding the characterization of HPLC stationary phases, they are only applicable to the samples in the dry state. In order to investigate the impact of polymerization time on the porous properties of wet monolithic columns, ISEC measurements of 200 jm I.D. poly(p-methylstyrene-co-l,2-bis(vinylphenyl)ethane) (MS/BVPE) capillary columns (prepared using a total polymerization time ranging from 45 min to 24 h) have been additionally evaluated (see Table 1.2 for a summary of determined e values). On a stepwise decrease in the time down to 45 min, the total porosity (St) is systematically increasing to about 30% in total (62.8% for 24 h and 97.2% for 45 min). This is caused by a simultaneous increase in the fraction of interparticulate porosity (e. ) as well as the fraction of pores (Cp). The ISEC measurements are in agreement with those of the MIP as well as BET analyses, as an increase in should be reflected in an increase in 8p and as the relative increase in the total porosity (caused by decreasing the polymerization time... 

Our initial attempts to separate the Cj and Dj isomers of 5 (G = 1) used octadecyl polysiloxane (ODS) as high performance liquid chromatography (HPLC) stationary phase and mixtures of acetonitrile/HjO or methanol/HjO as mobile phases. Under these classical reverse-phase conditions, the resulting efficiencies were extremely poor because of the low solubility of 5 (G = 1) in both mobile phases. By contrast, mixed mobile phases which contained acetonitrile (ACN) with some percentages of a cosolvent such as tetrahydrofuran (THF) substantially improved... 

Reta, M., Carr, R W., Sadek, P. C., and Rutan, S. C., Comparative study of hydrocarbon, fluorocarbon and aromatic bonded RP-HPLC stationary phases by linear solvation energy relationships. Anal. Chem., 71, 3484-3496, 1999. 

Development of new HPLC stationary phases is ongoing. A multimodal phenylpropanolamine-coated silica column was recently prepared and applied to the quantitation of ascorbic acid in orange juice and the separation of ascorbic acid from its epimer, isoascorbic acid (Table 28) (199). Other examples include new bonded phases and columns made of continuous porous rods that permit fast chromatography with low backpressure and high resolution (200-202). 

Traditional (i.e., non-MIP) SPE sorbents are similar to HPLC stationary phases. The advantages of many of these materials are that they are widely available, well characterized, have high binding capacity, and show linear adsorption behavior. One may observe that just a few types are used in the majority of sample preparations, i.e., these materials are quite generic, and it is the wash and elution step which is varied according to the application. The generic nature of these materials is also a drawback because it reflects their limited selectivity. 

Mi ne, M., Minato, K., Kitamura, Y., Okazaki, K., Iwado, A., Akizawa, H., Haginaka, J., Motohashi, N., and Saito, Y. (2004) Electron Interaction of PAHs with Anion-exchange Silica Gels Modified with Anionic Metal-porphine and -Phthalocyanine Derivatives as HPLC Stationary Phase for Preparative Column in Organic Solvents, Talanta 63, 1035-1038. 

The following table provides the most commonly used bonded phase modified silica substrates in solid phase extraction.1 Additional information on many of these materials can be found in the More Common HPLC Stationary Phases table in the HPLC chapter in this book. 

More Common HPLC Stationary Phases (continued)... 

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