The Chromatographie Process

The chromatography process is monitored visually. The exact volume of eluant added to the column will vary depending on sample concentration and composition. 

For a localized adsorption the concentration of the adsorption sites has to be taken into account [4], In addition, a reversible change of the chemical state of the adsorbate in the chromatography process has to be considered, e.g. dissociative and substitutive adsorptions as described in Part II of this chapter (Part n, Section 2.3, Equations 52-54). The reaction enthalpy and entropy has to be introduced into the calculations [5-8] as well as the... 

Very often, one of the tested mixtures can be used to perform the separation, or a few additional experiments using the same solvent combination have to be performed to fine-tune the chromatography process. 

Several characteristics of chromatography impact on the microdialysis experiment. The chromatography process inherently dilutes the sample. If we assume that a typical microdialysis experiment will involve a perfusion rate of 1 lL/min, with sampling for 5 min, 5 4L of sample will be obtained for the assay. A typical analytical column (15 cm X 4.6 mm) with a mobile-phase flow rate of 1 mL/min may have a peak width of 30 sec and would correspond to a 500-... 

Fig. 5 illustrates a physical model of the chromatography process. Initially, there is a dynamic equilibrium of molecules between the phases. Then, one phase is moved relative to the other with an average velocity, v. In the stationary phase, molecules do not move while in the mobile phase, molecules move with a velocity equal to v. Provided that the interphase mass transfer rate is fast relative to the flow rate of the mobile phase, the time-average distribution of a molecule between the phases is statistically equal to the equilibrium distribution as determined by the distribution constant. 

The frontal chromatographic analysis of a molecular-imprinted column allows not only the determination of adsorption energies and saturation capacities [65,66], but also determination of kinetic data for the association/dissociation process involved in the interaction between template and imprinted binding site, and mass transfer data for the chromatography process itself [67-69]. 

If a surfactant solution with concentration >cmc is applied to the chromatography column in an eluant as pure solvent (without any added surfactant), the monomer-micelle equilibrium is driven towards monomer due to dilution during the chromatography process. This system under dynamic conditions has been investigated by various investigators [45,48,52]. 

Susceptibility of the sample to shear damage should be considered. Most proteins can be so damaged, and although the chromatography process usually employs a single pass, in contrast for example to ultrafiltration, it may nevertheless be desirable to select low-shear pumps. Lobe and peristaltic pumps have lower shear characteristics than gear or centrifugal pumps. 

In the continuous process for producing phosphatidylcholine fractions with 70—96% PC at a capacity of 600 t/yr (Pig. 5) (16), lecithin is continuously extracted with ethanol at 80°C. After separation the ethanol-insoluble fraction is separated. The ethanol-soluble fraction mns into a chromatography column and is eluted with ethanol at 100°C. The phosphatidylcholine solution is concentrated and dried. The pure phosphatidylcholine is separated as dry sticky material. This material can be granulated (17). 

Gas chromatography (gc) has been used extensively to analyze phenoHc resins for unreacted phenol monomer as weU as certain two- and three-ring constituents in both novolak and resole resins (61). It is also used in monitoring the production processes of the monomers, eg, when phenol is alkylated with isobutylene to produce butylphenol. Usually, the phenoHc hydroxyl must be derivatized before analysis to provide a more volatile compound. The gc analysis of complex systems, such as resoles, provides distinct resolution of over 20 one- and two-ring compounds having various degrees of methylolation. In some cases, hemiformals may be detected if they have been properly capped (53). 

Gel-permeation chromatography studies of epoxy resins prepared by the taffy process shown n values = 0, 1, 2, 3, etc, whereas only even-numbered repeat units are observed for resins prepared by the advancement process. This is a consequence of adding a difunctional phenol to a diglycidyl ether derivative of a difunctional phenol in the polymer-forming step. 

In the development of new products, optimization of the fermentation medium for titer only often ignores the consequences of the medium properties on subsequent downstream processing steps such as filtration and chromatography. It is imperative, therefore, that there be effective communication and understanding between workers on the upstream and downstream phases of the produc t development if rational trade-offs are to be made to ensure overall optimahty of the process. One example is to make the conscious decision, in collaboration with those responsible for the downstream operations, whether to produce a protein in an unfolded form or in its native folded form the purification of the aggregated unfolded proteins is simpler than that of the native protein, but the refolding process itself to obtain the product in its final form may lack scalabihty. 

Factors to be considered in maldng the selection of chromatography processing steps are cost, sample volume, protein concentration and sample viscosity, degree of purity of protein product, presence of nucleic acids, pyrogens, and proteolytic enzymes. Ease with which different types of adsorbents can be washed free from adsorbed contaminants and denatured proteins must also be considered. 

Isolation procedures for many biochemicals are based on chromatography. Practically any substance can be selected from a crude mixture and eluted at relatively high purity from a chromatographic column with the right combination of adsorbent, conditions, and eluant. For bench scale or for a small pilot plant, such chromatography has rendered alternate procedures such as electrophoresis nearly obsolete. Unfortunately, as size increases, dispersion in the column ruins resolution. To produce small amounts or up to tens of kilograms per year, chromatography is an excellent choice. When the scale-up problem is solved, these procedures should displace some of the conventional steps in the chemical process industries. 

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