Size desalting

Gel filtration/size exclusion Size Desalting, large molecules... 

Electrodialysis Cation and anion exchange membranes Electrical potential gradient Electrical charge of particle and size Desalting of ionic solution... 

Sephadex. Other carbohydrate matrices such as Sephadex (based on dextran) have more uniform particle sizes. Their advantages over the celluloses include faster and more reproducible flow rates and they can be used directly without removal of fines . Sephadex, which can also be obtained in a variety of ion-exchange forms (see Table 15) consists of beads of a cross-linked dextran gel which swells in water and aqueous salt solutions. The smaller the bead size, the higher the resolution that is possible but the slower the flow rate. Typical applications of Sephadex gels are the fractionation of mixtures of polypeptides, proteins, nucleic acids, polysaccharides and for desalting solutions. 

Interleukin (from human source). Purified using lyophilisation and desalting on a Bio-Rad P-6DC desalting gel, then two steps of HPLC, first with hydroxylapatite, followed by a TSK-125 size exclusion column. [Kock and Luger J Chromatogr 296 293 7984 ]... 

Irrespective of the development of media, many of the traditional media are successfully defending their position. This is due to their hydrophilic nature, preserving biological function of the separated molecules, but also the fact that columns may be prepared easily and, finally, some of the classical media, e.g., Sephadex, have a selectivity that is so far unsurpassed and therefore very fit for use. Intersting enough, Sephadex is still the premiere gel filtration medium for desalting due to the optimal pore size and particle size of this medium (see Section II,C). 

Size exclusion is basically employed for three different applications. One is the original one, i.e., desalting or separation of solutes differing more than a decade in molecular size. Another application is fractionation where the size differences are smaller, i.e., typically a factor of two to five. The third application is the determination of the molecular weight(s) of a sample. 

The selectivity of a gel, defined by the incremental increase in distribution coefficient for an incremental decrease in solute size, is related to the width of the pore size distribution of the gel. A narrow pore size distribution will typically have a separation range of one decade in solute size, which corresponds to roughly three decades in protein molecular mass (Hagel, 1988). However, the largest selectivity obtainable is the one where the solute of interest is either totally excluded (which is achieved when the solute size is of the same order as the pore size) or totally included (as for a very small solute) and the impurities differ more than a decade in size from the target solute. In this case, a gel of suitable pore size may be found and the separation carried out as a desalting step. This is very favorable from an operational point of view (see later). 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

Hyperfiltration (Reverse Osmosis) is a form of membrane distillation or desalination (desalting) operating with membrane pore sizes of perhaps 1 to 10 Angstrom units. The various individual RO component technologies have improved tremendously over the last 20 to 25 years, and resistance to fouling and permeate output rates have benefited. Nevertheless, all RO plants remain susceptible to the risk of fouling, and adequate pretreatment and operation is essential to minimize this problem. 

Purify the derivatized dendrimer using gel filtration (size exclusion chromatography) on a desalting column or through use of ultrafiltration spin-tubes (for G-4 and above). For smaller dendrimers, the derivatives may be purified by repeated precipitation from a meth-anolic solution by addition of ethyl acetate, dioxane, or benzene. The SPDP-dendrimer may be dried by lyophilization (if in water or buffer) or by solvent evaporation in vacuo (if the precipitation method was used). 

Purify the biotin-dendrimer using size exclusion chromatography on a desalting matrix or by use of ultrafiltration (e.g., centrifugal concentrators). 

Purify the derivative by gel filtration using a PBS buffer or another suitable buffer for the particular protein being modified. The use of a desalting resin with low exclusion limits work well. To obtain complete separation, the column size should be 15-20 times the size of the applied sample. Fluorescent molecules often nonspecifically stick to the gel filtration support, so reuse of the column is not recommended. 

Purify the thiolated antibody by gel filtration using a desalting resin. Perform the chromatography using 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, containing lOmM EDTA as the buffer. To obtain efficient separation between the reduced antibody and excess reductant, the sample size applied to the column should be at a ratio of no more... 

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