Column packings surface characteristics

Liquid holdup is a function of liquid flow rate and column pressure drop. Two types of holdup have been defined. Static holdup is the volume of liquid per volume of packing that remains after gas and liquid flows are stopped and bed has drained. Static holdup depends on packing surface characteristics. The second type is operating holdup that is the volume of liquid per volume of packing that drains out of the bed after gas and liquid flows have been stopped. The gas flow rate has little effect on holdup below loading. 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

Phase separation (precipitation) of a polymer strongly depends on all its molecular characteristics. On the one hand, this allows very efficient separations in polymer HPLC utilizing phase separation and re-dissolution processes [20]. On the other hand, due to complexity of phase separation phenomena, the resulting retention volumes of complex polymers may simultaneously depend on several molecular characteristics of separated macromolecules. This may complicate interpretation of the separation results. Both precipitation and redissolution of most polymers is a slow process. It may be affected by the presence of otherwise inactive surface of the column packing. Therefore, the applicability and quantitative control of the phase separation phenomena may be limited to some specific systems of polymer HPLC. 

Proteins and antibodies are natural substrates for affinity columns because of the nature of the enzyme recognition site and the antibody-antigen interaction sites. They have a three-dimensional shape and electrical charge distributions that interact with only specific molecules or types of molecules. Once these substrate sites are identified, molecules can be isolated or synthesized with the key characteristics and used to build affinity supports. These substrates are often bound to a 6-carbon spacer so that they protrude farther away from the packing surface toward the mobile phase and are therefore more available. Certain natural and synthetic dyes have been found to serve as substrate mimics for a class of enzymes call hydrogenases and have been used to build affinity columns for their purification. 

More than 90% of the column packings are based on silica gel and its bonded phases [1], The primary reasons for its widespread use is because of high surface area and porosity, easy preparation, adjustable polarity, and good mechanical strength. Silica gel optimized for chromatography should have the characteristics listed in Table 5.1 [2]. 

Since the pioneering work of Knox et al. on CEC [9,10], porous silica particles have been used as the column packing material in the majority of research studies and applications. Porous silica has a number of characteristics that make it suitable for use in CEC. These are a large surface area, a high surface potential at moderate pH values, which allows the generation of a high EOF, and the commercial availability of materials with various surface chemistries. However, other support materials, such as polymeric phases [11] and alternative inorganic base materials [12], are also applicable in CEC. 

Kohler and Kirkland noted that for chromatographic column packings analyzed by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and chromatographic behavior, silica appears to possess associated and isolated surface silanol groups [ I9. The associated silanol (possessing weak-acid characteristics) is identified with a silanol hydrogen-bonded to its nearest neigh-... 

Silica (Si02) is the dominant support material, with excellent physical and chromatographic performance.1,5 Columns packed with unbonded silica are rarely used for analytical purposes due to the strong adsorptive characteristics. Silanol groups (Si-OH) found on silica surfaces are typically bonded with monochlorosilanes to create a hydrophobic liquid-like stationary phase for reversed-phase applications.1,12 Unreacted or residual silanols remaining after the bonding step are further reacted with a smaller silane (end-capped) to reduce the number of these adsorptive sites (Figure 3.4). One limitation of... 

Another application of pulse chromatographic methods is in studies of the kinetics of isotope-exchange reactions [75]. A deuterium-labelled compound was formed as a pulse of a volatile compound (reagent) passed through a column packed with Gas-Chrom A with 10% of Carbowax 6000 and 10% of KO H applied on its surface. The isotope exchange rate is a characteristic of the nature of the substance under investigation and is of... 

The structural variables of silica column packings have been summarized by Unger (6), including pore characteristics, surface area, and surface modifications. He also has described a procedure for making prous silica microspheres of controlled pore size (667), by hydrolyzing ethyl orthosilicate in an emulsion system. 

GPC separations require an interaction-free phase system. The separation efficiency in GPC is proportional to the pore volume, which limits the decrease in column dimensions. In many cases, GPC analyses are performed on serial combinations of columns. In most applications, silica and surface-modified silica columns do not meet the requirements for GPC columns and polymeric packings are preferred. Important characteristics for GPC column packings are the inertness of the sorbent material (chemistry), the accessibility of the pore structure, a high pore volume, and fast diffusion (optimal mass transfer) [la). 

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