Chromatography gel

Gel chromatography, as a separation method, is based on the size of molecules, which in turn determines the extent of their diffusion into the pores of gel particles packed as a stationary phase. Large molecules are excluded from most of the pores, whereas small molecules can diffuse further into the stationary phase. 

smaller molecules take a longer time to move down the column and are retarded in terms of their emergence from the column. 

A chromatography column of 10 mm i.d. and 100 mm height was packed with particles for gel chromatography. The interparticle void fraction e was 0.20. A small amount of a protein solution was applied to the column and elution performed in an isocratic manner with a mobile phase at a flow rate of 0.5 cm min. The distribution coefficient A of a protein was 0.7. An elution curve of the Gaussian type was obtained, and the peak width W was 1.30 cm . Calculate the Hs value of this column for this protein sample. 

This technique is particularly valuable in the separation of molecules of similar polarity but of different sizes, traditional methods of chromatography being less effective in these circumstances. The separation depends not only on the size but also on the shape of the molecules. There are several aspects by which separation effected by this technique can be viewed and this type of chromatography is variously named according to the particular separation aspect stressed upon. Accordingly, this technique is called gel filtration, gel permeation, molecular sieve filtration, molecular sieve chromatography, size-exclusion or simply exclusion chromatography. 

The total permeation volume, Vt, is the sum of the volume outside the gel grains i.e. the interparticle volume (V0), the volume of the liquid inside the gel grains (V ) and the volume of the gel matrix (Vg), that is, 

V0 may be easily measured by applying to the system a molecular species completely excluded from the gel system. V is the product of multiplication of the number of grams (a) of dry xerogel and the solvent regain, Wr, which is the amount of solvent taken up by one gram of dry xerogel. 

It is possible to define a parameter in gel filtration analogous to the partition coefficient in liquid-liquid or gas-liquid chromatography. In this case the distribution coefficient, KD, relates to the fraction of inner volume that is accessible to a particular species. If Ve is the elution volume in which a particular molecular species is eluted, then 

For a molecular species completely excluded, Ve = V0 and KD is zero. For small solute molecules which can enter all the pores, KD = 1. Separation, therefore, occurs only where solute molecules obey the condition 0 Kd i. 

The stationary phase is a porous polymer matrix whose pores are completely filled with the solvent to be used as the mobile phase. The pore size is highly critical, since the basis of the separation is that molecules above a certain size are totally excluded from the pores, and the interior of the pores is accessible, partly or wholly, to smaller molecules. 

The components of the mixture thus emerge from the column in order of relative molecular mass, the larger first. Any compounds which are completely excluded from the gel will not be separated from each other, and similarly, small molecules which completely penetrate the gel will not be separated from each other. Molecules of intermediate size will be retarded to a degree dependent on their penetration of the matrix. If the substances are of a similar chemical type, they are eluted in order of relative molecular mass. 

Adsorption effects of the surface of the gel particles can usually be ignored, and thus gel chromatography can be looked upon as a kind of partition chromatography. The liquid stationary phase is the liquid within the gel matrix, and the mobile phase is the flowing eluant which Alls the rest of the column. We have, in other words, a partition column where the two liquid phases, mobile and stationary, are of the same composition. 

The model proposed above suggests that the differential exclusion of the solute molecules was achieved on the basis of their hydrodynamic volumes, that is, their size and shape. This model has been extended by Porath [28], who considered the pores in dextran gels to be conical in nature. A further approach [29], which considers the gel to be composed of randomly arranged rigid rods, shows good correlation between the molecular radius and retention volume of the solute. 

Gel chromatography was originally used for separation of biological materials, because the earliest gel media, cross-linked dextrans, were suitable for use only with an aqueous system. In 1964 [26], cross-linked polystyrene gels suitable for use with organic solvents were first produced, and this made possible the extension of gel chromatography to the separation and characterisation of synthetic polymers. Application of the method, not only to a 

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I2O5 (400 mg 1.20 mmol) was added to a solution of a cycloalka[b]indole (l.OOmmol) in 80% aqueous THF (25 ml). The mixture was stirred at room temperature and the solvent removed in vacuo. The residue was extracted into EtOAc and the extract washed with water, 5% NaS203, saturated NaHCOj and brine and dried over Na2S04. The solvent was evaporated and the residue purified by silica gel chromatography. 

A solution of l-methylpyrano[4,3-b]indol-3-one (1 mmol) and methyl vinyl ketone (5 ml) in toluene (5 ml) containing 5% Pd/C (40 mg) was heated for 48 h in a sealed tube at 110°C. The reaction mixture was evaporated in vacuo and the residue purified by silica gel chromatography to give the product in 80% yield. 

N-Benzoyl L-leucylglyclne ethyl ester (3). N-Benzoy-L-leucine 1 (0.235 g, 1 mmol) and glycine ethyl ester hydrochloride 2 (0 1534 g, 1.1 mmol) in OMF (10 mL) under stirring vyas treated with diethylphosphoryl cyanide 3 (0.179 g, 1.1 mmol) In OMF at 0°C, followed by the addition of triethylamine (0.212 g, 2 1 mmoO. The mixture was stirred for 30 min at 0°C and 4 h at 20 C The reaction mixture was diluted with PhH-EtOAc, washed with 5% HCI, water, 5% NaHCOs solution and bnne. Evaporation of the solvent gave crude 4 which after sIDca gel chromatography afforded 0.271 g of 4 (66%) (pure L), mp 1S8-160°C. 

R SiBr, trace MeOH. Methoxymethyl ethers are stable to these cleavage conditions. Methoxymethyl esters are unstable to silica gel chromatography, but are stable to mild acid (0.01 N HCl, EtOAc, MeOH, 25°, 16 h)." ... 

Perylene [198-55-0] M 252.3, m 173-214°. Purified by silica-gel chromatography of its recrystd picrate. [Ware J Am Chem Soc 83 4374 1961.] Crystd from benzene, toluene or EtOH and sublimed in a flow of oxygen-free nitrogen. [Gorman et al. J Am Chem Soc 107 4404 1985 Johansson et al. J Am Chem Soc 109 7374 1987.]... 

Sodium taurocholate [145-42-6] M 555.7. Purified by recrystn and gel chromatography using Sephadex LH-20. 

Size exclusion HPLC has many other common names, such as gel permeation, gel filtration, steric exclusion, molecular sieve chromatography, or gel chromatography. These names all reflect the theoretical mode of action for this type... 

In general, the MIP ether is very labile to acid and silica gel chromatography, unless some TFA is used as part of the eluting solvent. The acid in the NMR solvent, CDCI3, is sufficient to cleave the MIP ether. 

To a stirred solution of 2,3-dimethyl indole (6, 1.45 g, 10 mmol, 1.0 equiv) and tetra-n-butylammonium sulfate (3.40g, 10 mmol, 1.0 equiv) in chloroform (150 mL) was added potassium hydroxide (50% aqueous solution, 20 mL) over 30 minutes. The stirring was continued for six hours, at which time the mixture was extracted with chloroform, the chloroform-water mixture was washed with water, and the organic layer concentrated. Silica gel chromatography provided 2,4-dimethyl-3-chloroquinoline (7, 1.52 g, 79% yield). 

After anilide 30 (1.12 g, 4.46 mmol) is hydrolized in 6 M HCl at 100 °C (by TLC analysis), toluene (5 mL) is added and then aldehyde 5 (0.74 mL, 8.92 mmol) is added dropwise at the same temperature. The reaction was stirred for 2 h and then cooled to room, temperature. The aqueous layer is removed and neutralized with aqueous NaOH to afford 31 as a crystalline solid. The crude product is purified by silica gel chromatography (hexanesiethyl acetate, 5 1) to give 31 (802 mg, 70%) as colorless crystals, mp 103 °C. 

The structure of the products were characterized by two detector gel-chromatography (recractometer and UV). The direction of the alkylation reaction of toluene with EC was changed depending on the reaction condition as follows if the ratio of toluene-EC is 1 5 mol, the alkylation reaction is toward obtaining the PEC at 273 K and if the ratio is reverse at 333 K, the reaction undergone to obtain epoxy toluene oligomer is shown in Fig. 1. 

Extraction and purification (Shimomura, 1991b). The luciferin precursors PS-A and PS-B are extracted from the dried fruiting bodies of Panellus stipticus (5 g) with methanol, and the extract is evaporated under reduced pressure to remove most of the methanol. The residue (pH 6.3) is diluted with a small amount of water and extracted with ethyl acetate, and the extract is evaporated to dryness. The precursors in the residue are purified by three steps of silica gel chromatography and one step of HPLC, while monitoring the absorbance of eluate at 220-230 nm. The procedure is summarized below ... 

The acid-catalyzed reaction of enol ethers 2 (X = OR) and enamines 2 (X = NR2) to form y-lactol derivatives proceeds with great ease even on silica gel chromatography. Vinyl sulfides 2 (X = SR) or vinyl chlorides 2 (X = Cl) are difficult to hydrolyze. 

Separation of the 4,5-anti- and the 4,5-. >, -diastereomers by silica gel chromatography is often extremely facile due to the lower polarity of the latter caused by the higher tendency for intramolecular hydrogen bridging97. 

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