Hydrodynamic column

Figure 7.3 Sketch illustrating the two kinds of CCC columns. (Top) Hydrodynamic columns contain a long coiled tube rotating in a planetary way that creates a succession of mixing and decantation zones always having the two liquid phases in contact. (Bottom) In hydrostatic columns, the stationary phase is contained in channels interconnected by ducts in which there is only mobile phase.

In hydrodynamic columns, there are spools of coiled tube that rotate on themselves and around a central axis. These combined rotations create a planetary motion with a highly variable centrifugal field that produces mixing zones followed by decanfafion zones (Figure 7.3). The stationary phase is partly retained inside the coils if fhe mobile phase is flown the right way. The coil rotation produces an Archimedean force that pushes the liquid phases toward one end of the coil called the head (higher pressure). 

The study of the IL-rich upper aqueous phase retention of fhe [C4CiIm] CI-K2HP04-water ATPS shows that hydrostatic colunms are able to retain liquid systems poorly retained by hydrodynamic CCC colunms. A few frials were done using the IL-rich phase as the mobile phase pushed in the ascending way. An acceptable retention of the phosphate-rich aqueous phase (72% at 1 mL/min and 900 rpm) was obtained with the hydrostatic colunm when no retention (S/ = 0%) could be obtained in all conditions with the hydrodynamic column. 

Light scattering coulter counter Particle size distribution, volume average particle diameter, number average particle diameter Stability of packed bed, hydrodynamic column properties, column performance... 

The use of an amperometric detector is emphasized in this experiment. Hydrodynamic voltammetry (see Chapter 11) is first performed to identify a potential for the oxidation of 4-aminophenol without an appreciable background current due to the oxidation of the mobile phase. The separation is then carried out using a Cjg column and a mobile phase of 50% v/v pH 5, 20 mM acetate buffer with 0.02 M MgCl2, and 50% v/v methanol. The analysis is easily extended to a mixture of 4-aminophenol, ascorbic acid, and catechol, and to the use of a UV detector. 

An interesting outgrowth of these considerations is the idea that In r versus K or Vj should describe a universal calibration curve in a particular column for random coil polymers. This conclusion is justified by examining Eq. (9.55), in which the product [i ]M is seen to be proportional to (rg ), with r = a(rg 0 ) - This suggests that In rg in the theoretical calibration curve can be replaced by ln[r ]M. The product [r ]M is called the hydrodynamic volume, and Fig. 9.17 shows that the calibration curves for a variety of polymer types merge into a single curve when the product [r ]M, rather than M alone, is used as the basis for the cafibration. 

Rizzuti et al. [Chem. Eng. Sci, 36, 973 (1981)] examined the influence of solvent viscosity upon the effective interfacial area in packed columns and concluded that for the systems studied the effective interfacial area a was proportional to the kinematic viscosity raised to the 0.7 power. Thus, the hydrodynamic behavior of a packed absorber is strongly affected by viscosity effects. Surface-tension effects also are important, as expressed in the work of Onda et al. (see Table 5-28-D). 

The column diameter is normally determined by selecting a superficial velocity for one (or both) of the phases. This velocity is intended to ensure proper mixing while avoiding hydrodynamic problems such as flooding, weeping, or entrainment. Once a superficial velocity is determined, the cross-sectional area of the column is obtained by dividing the volumetric flowrate by the velocity. 

The elution volume of a molecule in HPSEC is determined by its hydrodynamic size and the pore size of the column packing. In setting up an HPSEC experiment, the chromatographer must match the pore size of the column to the molecular size range of the sample. 

Proteins are separated on Zorbax GF columns based on their hydrodynamic size, which may be related to the proteins molecular weights (Fig. 3.10). Under ideal conditions, two proteins whose molecular sizes differ by a factor of 2 can be baseline separated. 

FIGURE 3.10 Under ideal conditions, proteins are separated on Zorbax GF columns based on their hydrodynamic size. 

Gels made in this way have virtually no usable porosity and are called Jordi solid bead packings. They can be used in the production of low surface area reverse phase packings for fast protein analysis and in the manufacture of hydrodynamic volume columns as well as solid supports for solid-phase syntheses reactions. An example of a hydrodynamic volume column separation is shown in Fig. 13.2 and its calibration plot is shown in Fig. 13.3. The major advantage of this type of column is its ability to resolve very high molecular weight polymer samples successfully. 

It is surprising to note that for all four columns there is good agreement between the M determined in water and in water/methanol, despite the fact that PEO and polyvinylpyrrolidone have different hydrodynamic volumes in these two mobile phases and that the column packings may swell or shrink differently in these two mobile phases. 

Solvent can affect separation in two different ways. Because water is a better solvent for these four columns than water/methanol, based on the swelling or void volume of the columns in Table 17.9, the separation should be better in water than in water/methanol. The relative viscosity of a 0.5% PEO standard from Aldrich (Lot No. 0021kz, MW 100,000) in water and in water/methanol with 0.1 M lithium nitrate is 1.645 and 1.713, respectively. This indicates that the hydrodynamic volume of PEO in water is smaller than in water/methanol. The difference in hydrodynamic volume between two PEO standards should also be larger in water/methanol than in water. Hence, the separation for PEO should be better in water/methanol than in water. The results in Table 17.8 indicate that separation efficiency is better in water than in water/methanol... 

Several factors can contribute to the difference in retention times for PEO in different mobile phases the viscosity of a mobile phase, the hydrodynamic volume of a PEO, and the swelling or void volume of a column. Shodex and TSK columns should swell more in water than in water/methanol, and PEO should therefore come out later in water than in water/methanol. PEO should also elute later in water than in water/methanol because water/methanol is a better solvent for PEO than water. The viscosity of the 50 50 water/methanol mobile phase is higher than the viscosity of water. PEO should therefore elute later in water/methanol than in water due to the difference in viscosity. The results in Table 17.9 indicate that the difference in retention time for PEO in water and in water/methanol depends more on the swelling of columns and the hydrodynamic volumes of PEO than the viscosities of mobile phases. 

Hydrodynamic volume refers to the combined physical properties of size and shape. Molecules of larger volume have a limited ability to enter the pores and elute the fastest. A molecule larger than the stationary phase pore volume elutes first and defines the column s void volume (Vo). In contrast, intermediate and smaller volume molecules may enter the pores and therefore elute later. As a measure of hydrodynamic volume (size and shape), SE-HPLC provides an approximation of a molecule s apparent molecular weight. For further descriptions of theoretical models and mathematical equations relating to SE-HPLC, the reader is referred to Refs. 2-5. 

The elution of such gels is an example not of size exclusion but rather of hydrodynamic fractionation (HDF). However, it must be remembered that merely being able to physically fit an insoluble material through the column interstices is not the only criterion for whether the GPC/HDF analysis of an insoluble material will be successful. A well-designed HDF packing and eluant combination will often elute up to the estimated radius in Eq. (5), but adsorption can drastically limit this upper analysis radius. For example, work in our laboratory using an 8-mm-bead-diameter Polymer Laboratories aqueous GPC column for HDF found that that column could not elute 204 nM pSty particles, even though Eq. (5) estimates a critical radius of —1.5 jam. 

TTiis ion exclusion effect can sometimes be exploited beneficially. For example, by purposefully choosing a column with some carboxyl groups and a pH that ionizes them (greater than approximately 6.5), it may allow separation of a charged and an uncharged polymer that have the same hydrodynamic size. Alternatively, one may be able to fine-tune elution of a polymer by adjusting pH. 

Figure 22.3 shows such theoretical curves as well as experimental points of capillary columns with 0.6- to 1.4-/i.m radii. The separation ranges of these capillary columns are from 5 X 10 to 10. Most of the data points follow the modified DG model. With OTHdC, the molecular hydrodynamic size can be calculated. However, the separation range of a single capillary column is relatively narrow, only about 1.5 order of magnitudes. 

The larger macromolecules can be separated using larger particle size columns. However, the flow rate should be watched carefully. As the effective hydrodynamic size of the macromolecules may be reduced due to the deformation by shear (23). Figure 22.8 shows that the effective hydrodynamic size of a 12-15 X 10 MW polyacrylamide sample will not reach its maximum, or the size without shear, unless the flow rate is reduced to 0.01 ml/min. A... 

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