Column packings totally porous

FIGURE 16.3 Dependences of the polymer retention volume on the logarithm of its molar mass M or hydrodynamic volume log M [T ] (Section 16.2.2). (a) Idealized dependence with a long linear part in absence of enthalpic interactions. Vq is the interstitial volume in the column packed with porous particles, is the total volume of liquid in the column and is the excluded molar mass, (b) log M vs. dependences for the polymer HPLC systems, in which the enthalpic interaction between macromolecules and column packing exceed entropic (exclusion) effects (1-3). Fully retained polymer molar masses are marked with an empty circle. For comparison, the ideal SEC dependence (Figure 16.3a) is shown (4). (c) log M vs. dependences for the polymer HPLC systems, in which the enthalpic interactions are present but the exclusion effects dominate (1), or in which the full (2) or partial (3,4) compensation of enthalpy and entropy appears. For comparison, the ideal SEC dependence (Figure 16.3a) is shown (5). (d) log M vs. dependences for the polymer HPLC systems, in which the enthalpic interactions affect the exclusion based courses. This leads to the enthalpy assisted SEC behavior especially in the vicinity of For comparison, the ideal SEC dependence (Eigure 16.3a) is shown (4). 

Totally porous particles of relatively large particle sizes were widely used in low pressure liquid chromatography for many years. These column packings had good sample capacity but only limited efficiency accompanied by long separation times, due to the large size and unfavorable size distribution of the particles and the presence of relatively deep pores within the particles through whick sample molecules diffused in and out of very slowly. 

One problem is how to optimize throughput (analysis time) without losing peak capacity. Different approaches have been suggested and led to different developments by instrument and column manufacturers. This section will concentrate on the usage of totally porous particle columns for chromatographic separation only. Alternatives are monolithic columns9 and shell packing materials such as Halo or Poroshell.10-13... 

The retention factor, k, is the basic value in chromatography, and is related to the void volume (dead volume). The void volume is the space inside the column, where no retention of solutes has occurred and can be measured on a chromatogram, as shown in Figure 1.3. The void volume is about half the total volume of the column when it is packed with porous stationary phase materials. In practice, the effective void experienced by the analyte is smaller because the molecular mass of the analyte is usually much greater than that of the eluent molecule. In a model of porous stationary phase material, the pores can be represented as V-shape valleys (Figure 3.8), where region a is a support, such as... 

The experimental of peptides or proteins depend more on the type of the column than on the gradient program and are constant within % at various gradient times. The variance in the (p is lower as the size of molecules increases. The values of (p are slightly higher with totally porous particles than with columns packed with superficially porous, nonporous particles and monolithic columns [97]. 

The use of column with superficially porous packing materials based on silica particles with nonporous cores is the most recently reported strategy for improving chromatographic performance. This technology, originally developed by Kirkland in the 1990s to limit diffusion of macromolecules into the pores [85], became commercially available in 2007 [86], In comparison with totally porous particles of similar diameters, the both A and C term of the Van Deemter curve are reduced [87, 88],... 

Fig. 4.7. Separation of 14 explosives in a 75 pm l.D. capillary 21 cm (total length of 34 cm) column packed with 1.5 pm non-porous reverse-phase silica particles. The mobile phase consisted of 20% methanol, 80% 10 mM MES, and 5 mM SDS separation voltage of 12 kV. Reprinted from ref. [69] with permission. Copyright American Chemical Society 1998.

Equation (8-3) is used to determine k values for experiments A-C, as indicated in the table. The Vr values for the dyes in this experiment are the volumes corresponding to the centers of the red and blue bands. No value is measured for Em, however. The best method for obtaining a true value of Em is to inject on the column a compound that is very similar, in chemical retentivity to the mobile phase and that responds to the detector. Such a compound would be unretained by the stationary phase and would elute after passing through the volume Em which is occupied by the mobile phase within the column. Due to the limitations of the apparatus used in these experiments, we will approximate Em- For a column filled with porous packing, the value of Em represents about 50% of the total empty column volume and can be estimated by the equation... 

FIGURE 10-1. Additivity of plates on a totally porous packing. Mobile phase 5% CH2C12 in isooctane (50% water saturated). Flow rate 2 mL/min. Detector UV, 254 nm, 0.5 AUFS. Sample Mixture of aromatics, 10 fiL. Column Porasil A, 2 mm ID x 122 cm. (Note Actual separation will depend upon quality of mobile phase and column packing.)... 

The porosity of particles suitable for packing HPLC columns depends on the size of molecules to be separated. Totally porous particles with a pore size of 7-12 nm and specific surface area of 150-400 m"/g are suitable for the separation of small molecules, but wide-pore particles with a pore size of 15-100 nm and relatively low specific surface area (10-150 nr/g) are required for the separation of macromolecules to allow easy access to the interactive surface within the pores. Packings with perfusion particles contain very broad pores (400-800 nm) throughout the whole particle interconnected by smaller pores. The mobile phase flows through the pores in the particle, which minimises both band broadening and column backpressure (111. Perfusion materials have been designed especially for the separation and isolation of biopolymers. 

Totally porous particles are most frequently used in contemporary HPLC and are available in various diameters, pore sizes and surface areas. The particle size of the column packing should be minimised to decrease the contribution of the mass-transfer... 

The total porosity, Stot, is about 0.8 for many column packings of totally porous particles without chemical derivatization. Thus Vq can be obtained from ... 

E lie total volume P, of a column packed with a porous polymer or silica gel is given by... 

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