Bioseparation columns

The HPLC of large biomolecules such as proteins and DNA often requires specialized columns packed with wide-pore polymer or silica-based bonded phase with extra-low silanol activity.1215 Alternate approaches are pellicular materials or very small nonporous particles. Some of these columns are packed in PEEK or titanium hardware to allow the use of high-salt mobile phase and to prevent possible protein denaturing by metallic leachates. Further details on bio-separations and application examples are discussed in Chapter 7. 

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Liquid column chromatography is the most commonly used method in bioseparation. As shown in Figure 11.6, the adsorbent particles are packed into a column as the stationary phase, and a fluid is continuously supplied to the column as the mobile phase. For separation, a small amount of solution containing several... 

Separation by Chromatography 175 Table 11.1 Liquid column chromatography used in bioseparation. 

Nandakumar, M. P., and Mattiasson, B. (1999). Binding assays in heterogeneous media using a flow injection system with an expanded micro-bed adsorption column. Bioseparation 8, 237-245. 

Coffman, J. L., Roper, D. K., and Lightfoot, E. N. (1994). High-resolution chromatography of proteins in short columns and adsorptive membranes. Bioseparation 4, 183-200. 

The required product quality and therefore the value of the product to the consumer will also influence the choice of bioseparation technique. Usually a more efficient or specific bioseparation technique will have higher cost, and so it would obviously be uneconomic to consider a series of chromatography columns to treat a very low value waste water stream to remove some specific impurities. 

Chromatography is an effective bioseparation technique suitable for low-volume, high-value products such as pharmaceutical proteins. In chromatographic separations, an aqueous or organic solution containing the product is passed through a packed column containing a separation matrix. Differences in chemical or physical properties between the product and its impurities are exploited to achieve the separation. 

For biopharmaceuticals separation, we can also find glass or plastic material (cross-linked polymers like acrylic). Table 4.3 gives typical dimensions and operating pressures of glass and large-scale acrylic columns for low-pressure bioseparations or intermediate purifications that are characterized by a height to diameter ratio < 1. 

In case of large retention factors of the last eluting component, which happens very often in bioseparations, chromatographic processes require the means to change the composition of the mobile phase in order to control the elution rate from the column. The rate at which the mobile phase composition changes concurs precisely with the elution time for each peak for a given process (Figure 5.26). 

Manipulating adsorption strength is most readily achieved by variation of solvent composition (or column pressure in the case of SFC-SMB, see Section 5.2.6.2). However, there exist further options for implementing gradients. In particular, the use of salt and pH gradients is attractive in the context of bioseparations (see some of the... 

The chromatographic separation approach is often used to describe a method of running an IX column or an adsorption column. These are not considered here see Sections 4.13 and 4.11 and 4.12 respectively. Two uses of the chromatographic approach are considered here affinity or immunosorbent (that is similar to ad-sorption/IX but is usually applied to bioseparations) and size exclusion or gel chromatography, SEC. 

Very often, particularly in bioseparation, extraction is used to separate solutes from each other fBelter et al.. 1988 Frank etal.. 2008 Schiebel, 1978). In this situation we can use fractional extraction with two solvents as illustrated in Figure 13-5. In fractional extraction the two solvents are chosen so that solute A prefers solvent 1 and concentrates at the top of the column, while solute B prefers solvent 2 and concentrates at the bottom of the column. In Figure 13-5 solvent 2 is labeled as diluent so that we can use the nomenclature of Table 13-2. The column sections in Figure 13-5 are often separate so that each section can be at a different pH or temperature. This will make the equilibrium curve different for the two sections. It is also common to have reflux at both ends (Schiebel, 1978). 

Affinity Precipitation. All bioseparation processes include three stages preferential partitioning of target substance and impurities between two phases (liqnid-liqnid or liquid-solid), mechanical separation of the phases (eg, separation of the stationary and mobile phases in a chromatographic column), and recovery of the target snbstance from the enriched phase. Because smart polymers can undergo phase transitions, they could facilitate the second and the third stages of bioseparation processes. 

The tubing connecting the sample introduction unit, column, and detector consists of small-volume stainless steel capillaries with an inside diameter of 0.25 mm and an outer diameter of 1.25 mm (1/16 inch). For bioseparations that are sensitive to stainless steel, tubing made from polyethere-therketone (PEEK) is recommended. All connecting tubing, e.specially between sample introduction system and column, should have a small vol-... 

One of the newest developments in ROMP-based monohths for bioseparations is boronate affinity columns synthesized via Schrock-initiated ROMP of NBE and DMN-H6 with the porogenic solvents w-hexane and 1,2-dichloroethane. Functionalization was achieved via post-grafting of a boronate-containing monomer. These columns were used to separate cis-diol-based biomolecules, such as adenosine [116]. 

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