Separations Based on Size

The simplest physical property that can be exploited in a separation is size. The separation is accomplished using a porous medium through which only the analyte or interferent can pass. Filtration, in which gravity, suction, or pressure is used to pass a sample through a porous filter is the most commonly encountered separation technique based on size. 

Particulate interferents can be separated from dissolved analytes by filtration, using a filter whose pore size retains the interferent. This separation technique is important in the analysis of many natural waters, for which the presence of suspended solids may interfere in the analysis. Filtration also can be used to isolate analytes present as solid particulates from dissolved ions in the sample matrix. For example, this is a necessary step in gravimetry, in which the analyte is isolated as a precipitate. A more detailed description of the types of available filters is found in the discussion of precipitation gravimetry and particulate gravimetry in Chapter 8. 

Illustration of a dialysis membrane in action. In (a) the sample solution is placed in the dialysis tube and submerged in the solvent, (b) Smaller particles pass through the membrane, but larger particles remain within the dialysis tube. 

A method of separation that uses a semi-permeable membrane. 

A separation method in which a mixture passes through a bed of porous particles, with smaller particles taking longer to pass through the bed due to their ability to move into the porous structure. 

---

A form of capillary electrophoresis in which the capillary column contains a gel enabling separations based on size. 

As mentioned previously, Bibette [95] has developed a very elegant method for the purification of coarse, polydisperse emulsions to produce monodisperse systems. This technique is based on the attractive depletion interaction between dispersed phase droplets, caused by an excess of surfactant micelles in the continuous phase. A phase separation occurs under gravity, between a cream layer and a dilute phase since the extent of the separation increases with increasing droplet diameter, a separation based on size occurs. By repeating this process, emulsions of very narrow size distribution can be produced. 

Molecular exclusion chromatography is based on the inability of large molecules to enter small pores in the stationary phase. Small molecules enter these pores and therefore exhibit longer elution times than large molecules. Molecular exclusion is used for separations based on size and for molecular mass determinations of macromolecules. In affinity chromatography, the stationary phase retains one particular solute in a complex mixture. After all other components have been eluted, the desired species is liberated by a change in conditions. 

Although not a force, sieving is another mechanism by which SCpcL-rations can be achieved in chromatography. Probably sieving is not the best term to use, but it does denote that separations are made on the basis of the sizes of the sample molecules. In fact, in their most common form, chromatographic separations based on size are achieved by controlling the size of the pores in the stationary phase so that some (small) molecules... 

IMS is a relatively new technique in which ions are separated based on size and shape using an electric field. IMS was utilized by Dong et ah (2010) to separate all-trans -lycopene from cw-lycopene and all-irans -(3-carotene from cw-(3-carotene. Unfortunately, the various cis isomers could not be separated from each other using IMS alone. The authors provided evidence to suggest that cis/trans isomerization of carotenoids occur in-source (ESI positive mode was used in these experiments). Because of this isomerization, it does not appear likely that IMS will replace HPLC as a means of separating geometrical isomers of carotenoids in the near future (Dong et ah, 2010). 

Humic substances occur in every natural water sample which has been analyzed for their presence. The amount and composition of humic substances vary considerably from soils, surface waters, and groundwaters, but their ubiquity in water is without question. Humic substances in water occur as a size continuum ranging from dissolved through colloidal, to particulate phases. The dissolved phase is a predominant phase in most streams and is the phase emphasized in this chapter. The geochemical activity and reactivity of dissolved and particulate organic phases are thought to be of sufficient difference in magnitude to merit the separation based on size at 0.45 /xm. 

Molecular sieves are porous synthetic zeolites of some modified form ofNa 2(A102)i2(Si02),2 27H2O. The most common pore sizes are 0.5 and 1.3 nm. They separate based on size and can be reactivated by heating to 300 "C for 1 hour. 

What is the difference between agarose gels and polyacrylamide gels Agarose gel electrophoresis is mainly used for separating nucleic acids, although it can also be used for native gel separation of proteins. Acrylamide is the usual medium for protein separation. When acrylamide gels are run with the chemical SDS, then the proteins separate based on size alone. 

Recall Why is the order of separation based on size opposite for gel filtration and gel electrophoresis, even though they often use the same compound to form the matrix ... 

Reflect and Apply When proteins are separated using native gel electrophoresis, size, shape, and charge control their rate of migration on the gel. Why does DNA separate based on size, and why do we not worry much about shape or charge ... 

SEC. Another liquid chromatographic technique applied in biopharmaceutical characterization is SEC. In this technique, compounds are separated based on size and shape. In contrast to RP-HPLC, SEC has a low resolving power that has limited the application of the technique. The main application of SEC in pharmaceutical analysis is the determination of the native size of a protein and the quantihcation of protein aggregates. Typically protein-friendly conditions are employed in SEC that are expected not to affect the higher order structure of the protein. 

Gas separation based on size/shape exclusion has been achieved in a relatively large number of small pore frameworks. The 3D diamond-type framework [Mn°3(HCOO)6], for example, selectively adsorbs H2 over N2 and Ar at 78K and CO2 over CH4 at 195 K. Uptakes of the excluded gases N2, Ar and CH4 are almost zero due to their inability... 

The equipment for UF systems often looks very similar to RO systems although they operate at lower pressures. However, this similarity does not extend to the molecular level. Remeniber that RO membranes are nonporous and separate based on a solution-diffusion mechanism UF membranes are porous and separate based on size exclusion. Large molecules are excluded from pores in the thin membrane skin and thus, the large molecules are retained in the retentate. Small molecules fit into the pores and pass through to the permeate. Since there is usually a distribution of pore sizes, molecules within the range of pore sizes partially permeate and are partially retained. In a somewhat oversinplified picture, UF is cross-flow filtration at the molecular level. 

In a process used since 1984, a mixture of calcium hypochlorite dihydrate and larger sodium chloride crystals are formed by chlorination and then separated based on size and density. " Solutions of calcium hypochlorite and sodium chloride recycled from the process are mixed with lime to make a slurry of dibasic calcium hypochlorite and unreacted lime. Gravity sedimentation... 

Hollow fibers have proved to be useful to separate labeled hapten from the complex labeled hapten-Ab after a competitive on-line HPLC-immunochromato-graphic detection method [121]. First, hapten eluting from the HPLC column was made to react with Ab. Then, labeled hapten was added to react with the excess of Ab. Separation of free labeled hapten from labeled hapten-Ab was performed using a hollow-fiber module. The cross-flow membrane of this module allowed separation based on size, as opposed to a restricted-access media (RAM) column in which size and hydrophobicity account for separation [122]. No restriction on polarity and size of the label and no need for regeneration of the separation device arc characteristic of the use of hollow fibers. Although the method has been used for analysis of haptens, it could be used to analyze proteins as it has been shown that proteins of 40kDa can be separated from the corresponding protein-Ab complex if the appropriate hollow fiber is chosen. 

Separation based on size and charge differences between solutes techniques exploiting these differences are termed Capillary Zone Electrophoresis (CZE) or Free Solution Capillary Electrophoresis (FSCE)... 

---