Sample displacement chromatography

Both frontal and displacement chromatographies suffer a significant disadvantage in that once a column has been used, part of the sample remains on the column. The column must be regenerated before reuse. In elution chromatography all of the sample material is usually removed from the column... 

A sample similar to that shown in Figure 11.10 was used as the feed in displacement chromatography separations [36], For these experiments, the authors employed a longer (105 mm) column of the same diameter as the analytical column. Displacement chromatography on 10 mg of a melittin sample was carried out at two temperatures to demonstrate the advantage of higher temperature on mass transfer kinetics in the separation (Figure 11.11). 

Displacement chromatography is commonly used for preparative-scale separations, but, because of its focusing or concentrating effect, it also shows potential on the analytical scale, for example, for the concentration of minor components in complex mixtures.24,25 Operationally, displacement chromatography is similar to the step elution process, except that in the displacement process the mobile phase has a greater affinity for the stationary phase than for the sample components, and therefore the components are eluted ahead of the displacer front. The focusing effect of displacement chromatography is due to the fact that the concentration of the displacer determines the concentration of the product bands.26... 

When purifying oligonucleotides it is particularly useful to use sample self displacement chromatography since the required component of the mixture is generally the later eluting moiety. With this approach the column loading is increased to such a point that the more strongly retained component displaces the... 

Two case studies will be shown here to demonstrate the development of purification processes in both overload and resolution based separations 51]. The first example summarizes the purification of a synthetic peptide by overload chromatography, or more accurately described as sample self displacement chromatography The techniques applied to this separation are applicable to any molecule and can be applied to all modes of chromatography, with the exception of size exclusion chromatography. 

FIGURE 14 Purification of rHu-BDNF from its variants by displacement chromatography. (Barnthouse et a/.39) (A) Analytical chromatography of feed stock by high temperature reversed-phase acetonitrile gradient. Column 4.6 X 250 mm Vydac C4 sample 50 jj.% injection of rHu-BNDF. (B) Displacement of rHu - BDNF by protamine. Column 4.6 X 500 mm, POROS HS / M loading 20 mg / mL column volume mobile phase 50 mM phosphate buffer, pH 7.0, 500 m/W NaCI displacer 2 mM protamine sulfate. (C) High-temperature reversed-phase assay of three displacement fractions. Overlay of fractions from the early (fraction 16), intermediate (fraction 18), and late (fraction 31) parts of the displacement train. 

Hodges, R. S., Burke, T. W. L., and Mant, C. T. (1988). Preparative purification of peptides by reversed phase chromatography—sample displacement versus gradient elution modes. ]. Chromatogr. 444, 349-362. 

D. Displacement Chromatography and Sample Self-Displacement Chromatography... 

Additional results from AX purification of DMT-off synthetic oligonucleotides are discussed in the following section, based on the principles of displacement and sample self-displacement chromatography. 

FIGURE 10 Purification of a 8 mer phosphorothioate on AX with sample self-displacement chromatography. A 8 mer phosphorothioate. DMT-off crude was purified on an AX column (Q Hyper D F, BioSepra Corp., Marlborough, MA). Buffer A 50 mM NaOH buffer B 50 mM NaOH + 2.5 M KCI column 50 X 100 mm Waters AP-5 column flow rate 35 mL / min (107 cm / hr). Gradients for the two purification rounds are shown in the chromatograms. A pool for the first purification rounds was collected, the conductivity reduced by dilution with water and injected for the second purification. Feed purity by CGE 77%. Purity of the product pool after second cycle 98% by CGE. Purity by AX feed =71%, product pool after second cycle = 99.5%. 

The concentration depends only on that of the leading ion it is independent of the initial concentration in the sample. Therefore, ITP can act as an enrichment method, analogous to displacement chromatography and in contrast to zone electrophoresis and elution chromatography. The concentration in the steady state is adjusted to the value given in Eq. (2). If the concentration of the analyte species is lower in the initial sample, the higher steady-state concentration is established. This concentration is independent of the migration distance there is no dilution with a BGE as there is in capillary zone electrophoresis (CZE). 

Displacement chromatography is characterized by the introduction of a discrete volume of sample into the chromatographic column that has been previously equilibrated with a weak mobile phase, termed the carrier. This carrier is chosen so that the individual components of the sample (the solutes) are significantly retained by the stationary phase. The displacement is accomplished by following the sample with a new mobile phase containing some concentration of the displacer, a molecule with a higher affinity for the stationary phase than that of any of the solutes. The solutes are displaced from the stationary phase by the higher-... 

Experimental work of Kalasz et al. resulted in the statement of the characteristics and basic rules of displacement chromatography. They conceived properties of the fully developed displacement train, factors affecting displacement development, efficacy of separation, analysis of displaced fractions, determination of displacement diagrams from Langmuirian isotherms, as well as selection of the column, carrier, and displacer for displacement chromatography. Concentration of the sample is a particular feature of displacement chromatography. However, the displacer in the carrier is also definitely concentrated through the development of the displacement train. 

In displacement chromatography, the sample to be purified is injected in a large volume, or even continuously, into the CCC machine. The sample components have different affinities for the stationary phase in an exclusive way The component with a higher affinity for the stationary phase displaces another one with a lower affinity. Bands of pure components form. This method of using CCC offers the maximum throughput capability [1]. 

The pH-zone-refining CCC technique shares many unique features with displacement chromatography [6] and has several important advantages over the standard CCC technique such as (a) large sample-... 

In the late 1940s and early 1950s, the American Petroleum Institute (API) used displacement chromatography to fractionate samples of virgin crude oil and petroleum distillates to determine their content of paraffins, naphthenes, olefins, and aromatics and to isolate many pure compounds that were identified for the first time in crude oil or crude oil extracts [42]. In addition to working on the laboratory scale. 

In gradient elution or in displacement chromatography, the boxmdary condition given by Eq. 2.7 still applies to the sample components. However, the mobile phase contains an additive at a concentration which varies during the experiment. This additive is the strong solvent in gradient elution and the displacer in displacement chromatography. 

Figure 12.2 Influence of the sample size on the band profQes in displacement chromatography. Same parameters as in Figure 12.1b, except Q = 125 mg/mL and H =...

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