Eluent column chromatography

Anthrarufin [l,5-dihydroxy-9,10-anthraquinone] [117-12-4] M 240.1, m 280 (dec), pKj 9.90, pK 11.05. Purified by column chromatography on silica gel with CHCl3/Et20 as eluent, followed by recrystn from acetone. Alternatively recrystd from glacial acetic acid [Flom and Barbara J Phys Chem 89 4489 1985]. 

A mixture of 22 parts of 1 -ethyl-1,4-dihydro-5H-tetrazol-5-one,45 parts of 1 -bromo-2-chloro-ethane,26 parts of sodium carbonate,0.3 part of potassium iodide and 240 partsof 4-methyl-2 pentanone is stirred and refluxed overnight with water-separator. The reaction mixture is cooled, water is added and the layers are separated. The aqueous phase is extracted three times with dichloromethane. The combined organic phases are dried, filtered and evaporated. The residue is purified by column-chromatography over silica gel using trichloromethane as eluent. The pure fractions are collected and the eluent is evaporated, yielding 28.4 parts (80%) of 1-(2-chloroethyi)-4-ethyl-1,4-dihydro-5H-tetrazol-5-one as a residue. 

To this solution was added at one time the above-obtained ethyl acetate solution at -15°C, and the resulting mixture was stirred for 1 hour at -10°C to -15°C. The reaction mixture was cooied to -30°C, and water (80 ml) was added thereto. The aqueous layer was separated, adjusted to pH 4.5 with sodium bicarbonate and subjected to column chromatography on Diaion HP-20 resin (Mitsubishi Chemical Industries Ltd.) using 25% aqueous solution of isopropyl alcohol as an eluent. The eluate was lyophilized to give 7-[2-methoxyimino-2-(2-amino-1,3-thiazol-4-yl)acetamido] cephalosporanic acid (syn isomer) (1.8 g), MP 227°C (decomp.). 

To which a solution of manganese sulfate (15 g), 3.1 g of chromium trioxide, 72 ml of water and 3.5 ml of sulfuric acid was added. After stirring for 3.5 hours at 3°C, extracted with diethyl ether. The organic layer was washed with water, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using ethyl acetate-benzene (1 1) as eluent to give 2.35 g of the title compound. 

Removal of solvent from the extracts leaves a residue that is purified by dry-column chromatography.2 The residue is dissolved in 40 ml. of acetone in a 300-ml., round-bottomed flask, 30 g. of silica gel (Note 8) is added, and the acetone is removed with a rotary evaporator. The resulting solid mixture is placed on top of 360 g. of dry silica gel (Note 8) packed in flexible nylon tubing (Note 9), and the column is developed with 420 ml. of 10 1 (vjv) benzene-acetone. Approximately 150 ml. of solvent drips from the bottom of the column toward the end of development, and this eluent is collected in 25-ml. fractions and checked for product by thin layer chromatography (Note 10). The column itself is then cut into 2-cm. sections, the silica gel in each section is eluted with three 25-ml. portions of ethyl acetate, and the eluent from each section is analyzed by thin-layer chromatography (Note 10). Combination of all the product-containing fractions yields 1.2-1.5g. (40-47%) of the benzylated compound as an oil, n 1.6083 (Notes 11 and 12). 

Eluents for silica gel column chromatography toluene (Eluent 2) and toluene-acetone (19 1, v/v) (Eluent 3)... 

Solvent-partitioned mixtures are fractionated further by column chromatography involving Sephadex LH-20, silica gel, or a combination [19]. Quantitative recovery of constituents is often possible with Sephadex chromatography. With a MeOH/CHCl3 (1 1) eluent, pigmented and other polar material is removed in the early fractions, while the nonpolar isonitrile-related compounds elute later. 

A method recommended for adoption as official, first action (27). The method in which methimazole is separated from tablet excipients by column chromatography on Celite 545 with chloroform as the eluent and then quantitatively measured and identified by IR spectrophotometry. This method was studied collaborative-ly by 10 analysts average recoveries from two simulated and two tablets mixtures ranged from 96.6% + 1.0 to 101.1% + 0.9 (28). 

A number of minor components present in commercial neomycin have been separated by column chromatography on a carboxylic cation-exchange resin, Amberlite CG-50 99. The components were eluted from the resin with ammonium hydroxide solution. T.L.C. of the eluent fractions showed the presence of two previously unreported impurities which were then isolated on Dowex 1X2 and tentatively identified using NMR and mass spectrometry. 

The filtrate was concentrated using a rotary evaporator and the residue was purified by column chromatography on silica gel (eluent hexane-ethyl acetate, 30 1) to give (2S, 3R)-epoxychalcone (99%) as a colourless solid. 

Purification by flash column chromatography on silica (eluent petroleum ether-ethyl acetate, 2 1) gave a crystalline solid (S)-TV-(tert-butoxycarbonyl)-l-(4-methoxyphenyl)-2-hydroxyethylamine (296 mg, 74%). 

The residue was purified by silica gel column chromatography using n-hexane-ethyl acetate (3 1 —> 1 1) as an eluent to give as a white solid 3-benzyloxyamino-2-butanol (1.03 g, 92%) as a mixture of diastereomers. 

In column chromatography, the adsorbant is usually alumina but can be silica gel. Except that alumina tends to be basic and silica gel, acidic, I don t know why the former is used more often. Remember, if you try out an eluent (solvent) on silica gel plates, the results on an alumina column may be different. 

In column chromatography the mobile (moving) phase is a liquid that carries your material through an adsorbant. I called this phase the eluent, remember Here a gas is used to push, or carry, your vaporized sample, and it s called the mobile phase. 

Air bubbles are the nasties in HPLC work. They cause the same type of troubles as with wet-column chromatography, and you just don t want them. So there s usually a bubble trap (Fig. 112) before the eluent reaches the pump. This device is quite simple, really. Bubbles in the eluent stream rise... 

A novel development for HPLC is something called bonded reversed-phase columns, where the stationary phase is a nonpolar hydrocarbon, chemically bonded to a solid support. You can use these with aqueous eluents, usually alcohol-water mixtures. So you have a polar eluent and a nonpolar stationary phase, something that does not usually occur for ordinary wet-column chromatography. One advantage is that you don t need to use anhydrous eluents (very small amounts of water can change the character of normal phase columns) with reversed-phase columns. 

There are much better parallels to HPLC TLC or column chromatography. Vary the eluents in these techniques and you get widely different results. With a normal-phase silica-based column, you can get results similar to those from silica gel TLC plates. 

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