Analytical columns, particle sizes

To achieve the optimum reversed-phase LC separation, one needs to explore variables such as the analyte chemistry, mobile-phase composition (solvent type, solvent composition, pH, and additives), column composition, column particle size, and column temperature. For pharmaceutical analysis using mass spectrometry, the chemistry of an analyte is rarely changed beyond manipulation of the mobile phase pH, and even there options are limited. Volatile pH modifiers (buffers) are still preferred for LC-MS, and concentrations of these modifiers are kept low. Relatively simply mobile phases consisting of water, acetonitrile, and either formic acid (0.1% v/v), ammonium acetate (1-20 mM), or both have been common. 

Matrix (volume) Analytes Internal standard LOQ (ng/ml) Sample preparation Column (particle size, dimensions) Ionization and detection mode Reference... 

The column efficiency, backpressure and lifetime should be taken into account and compromised when selecting the best column particle size. Most often, porous particles with diameters of 5 pm are used in conventional analytical columns and 3 pm (exceptionally 2 pm) porous particles are usually used in short high-speed columns for... 

Mobile phase mass-transfer coefficient, CmU A quantity that affects band broadening and thus plate height nonlinear in solvent velocity u and influenced by the diffusion coefficient of the analyte, the particle size of the stationary phase, and the inside diameter of the column. 

The analytical capability of a SEC column is sometimes judged by the peak capacity, which is the number of unique species that can be resolved on any given SEC column. This number will increase with decreased particle size, increased column length, and increased pore volume. Because small particlesized medium generally has a lower pore volume and a shorter column length, peak capacities of ca. 13 for fully resolved peaks can be expected for high-resolution modern media as well as traditional media, (see Eig. 2.5). It was found that SEC columns differ widely in pore volume, which affects the effective peak capacity (Hagel, 1992). 

Column type Column size (mm) Theoretical plate number Particle size (pm) Pore size (A) Maximum flow rate (ml/min) Maximum pressure (kgf/cm ) Maximum temperature (°C) Exclusion limit Analytical column... 

Below the dotted line in Table I we list less fundamental differences between the two methods. Column lengths tend to be somewhat shorter in HPLC using small particle PB as a consequence of the high efficiencies that can be generated with the smaller particle sizes. For analytical scale HPLC, tube diameters of 3-4 mm are selected however, for preparative scale, tube diameters of 1 cm or above are not uncommon. 

Selection of columns and mobile phases is determined after consideration of the chemistry of the analytes. In HPLC, the mobile phase is a liquid, while the stationary phase can be a solid or a liquid immobilised on a solid. A stationary phase may have chemical functional groups or compounds physically or chemically bonded to its surface. Resolution and efficiency of HPLC are closely associated with the active surface area of the materials used as stationary phase. Generally, the efficiency of a column increases with decreasing particle size, but back-pressure and mobile phase viscosity increase simultaneously. Selection of the stationary phase material is generally not difficult when the retention mechanism of the intended separation is understood. The fundamental behaviour of stationary phase materials is related to their solubility-interaction... 

A RAM column functions through a size exclusion mechanism. Large biomolecules such as proteins are restricted from the adsorptive surfaces inside silica particles. Small analyte molecules are able to penetrate into the inner surfaces of the particles. As a result, protein molecules pass through the column rapidly and analytes of interest are retained on the adsorptive sites. Depending on the application, the analyte molecules are directed to MS for detection or transferred onto an analytical column for separation prior to MS detection. Detailed applications are discussed in a recent review.8... 

Where a, b, and c = van Deemter coefficients, dp = particle size of column, L = column length, Dm = diffusion coefficients of analytes, t = column dead time (depends on flow rate F), tg= gradient time (determines analysis time via tA = tg + t0), Ac = difference in concentrations of the organic modifier at the end and the beginning of the gradient (a continuous linear gradient is assumed), and B = slope of the linear relationship between the logarithm of the retention factor and the solvent composition. 

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