Gel Filtration Media

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). 

Finally, the construction should allow very flexible handling to cope with the various packing instructions recommended by the suppliers of the gel filtration medium. 

The packing method supplied by the manufacturer of the gel filtration medium may need to be revised according to the column being selected. It is therefore important to have an understanding about the basic principles governing the packing of chromatographic beds. 

Samples were fractionated by size on stainless steel columns packed with TSK-GEL ToyoPearl gel filtration medium, (Supelco Inc., Bellefonte, PA) in 50 mM NH4AC, pH 5.2. For HW-55 S (fractionation range for dextrans 1000-200,000 daltons) and HW-50 S (fractionation range for dextrans 500-20,000 daltons), the column was 10 x 500 mm and the flow rate was 1 ml/min. For HW-40 S (fractionation range for dextrans 100-7000 daltons),... 

Prepare a slurry containing a suitable amount of gel filtration medium in 50% (v/v) aqueous methanol containing 0.1 % (v/v) TFA, and pack a suitably sized column with this slurry according to the manufacturer s instructions. 

Sepharose CL-6B (Amersham Biosciences, Chalfont St. Giles, UK) or a similar gel filtration medium. 

In the time that is left, let us look at several examples of the use of a l c-detector Poetical situations. Figure 8 shows the resolution of two C-metabolites from each other in a mixture of naturally-occurring compounds in river water. This is an early stage purification step on BioGel P-2, a gel filtration medium. Each of these peaks is now ready to be purified further by chromatography in some other mode. 

Of the iron in the monoferric phytate-enriched bread, 70% was extracted into 1.2 M ammonium acetate, and the iron chromatographed as monoferric phytate on gel filtration medium. 

Gel filtration[91 is a key method in the purification of enzymes as well as biological macromolecules. It is reliable and simple as a separation technique without adsorption and interaction on gel filtration media. In gel filtration, the principle of separation is very simple, and macromolecules in solution are separated based on differences in their size as they pass through a column (Fig. 2-12). Large molecules pass through the stationary phase first while smaller molecules move about the gel filtration medium slowly. Gel filtration is also called molecular-sieve chromatog-... 

The dead volume at the inlet and outlet should be less than 0.1%. A sample volume of 0.5-5% of the bed volume is recommended for good resolution and depends on the gel filtration medium. The relationship between sample volume, medium and resolution is described however, the actual sample volume should be determined by experiment. A smaller sample size is not good for resolution. Up to 30% of the total bed volume can be applied for changing the buffer and salting out. 

Centricon 30 filtration units (Amicon) or Filtron Microsep 30 (Filtron) PDIO gel filtration medium (Pharmacia) 5(6)-carboxyfluoroscein-N-hydroxysuccinimide ester Boehringer cat. 10055089 stock solution, 10 mg/ml in DMSO Sulfo SMCC [sulfosuccinimidyl 4-(/V-maleimidomethyl)cyclohexane-l-carboxylate]. Pierce Chemical Company (cat. 22322), 20 mg/ml stock solution in 50 mM borate buffer, pH 7.6, freshly prepared... 

A gel filtration medium should possess the following characteristics ... 

The gel-filtration medium is normally packed into a stainless steel column characterized by an i.d. between 4 and 25 mm and a length between 300 and 600 mm. Four important factors in characterizing SEC packings are the average particle size, the particle size distribution, the average pore diameter, and the pore size distribution. The particle size and the particle size distribution have a significant influence on the column efficiency, which is most decisive in SEC, because the biopolymers are eluted isocraticaUy in a small elution window with... 

The experimentally determined relationship between solute size (Stokes radius or molecular weight) and the distribution coefficient is a fundamental characteristic of any gel filtration medium known as the selectivity curve. 

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