Eluting the Column

Purification of anthracene. Dissolve 0-3 g. of crude anthracene (usually yellowish in colour) in 160-200 ml. of hexane, and pass the solution through a column of activated alumina (1 5-2 X 8-10 cm.). Develop the chromatogram with 100 ml. of hexane. Examine the column in the hght of an ultra-violet lamp. A narrow, deep blue fluorescent zone (due to carbazole, m.p. 238°) will be seen near the top of the column. Immediately below this there is a yellow, non-fluorescent zone, due to naphthacene (m.p. 337°). The anthracene forms a broad, blue-violet fluorescent zone in the lower part of the column. Continue the development with hexane until fluorescent material commences to pass into the filtrate. Reject the first runnings which contain soluble impurities and yield a paraffin-hke substance upon evaporation. Now elute the column with hexane-benzene (1 1) until the yellow zone reaches the bottom region of the column. Upon concentration of the filtrate, pure anthracene, m.p. 215-216°, which is fluorescent in dayhght, is obtained. The experiment may be repeated several times in order to obtain a moderate quantity of material. 

Purification of Antibiotic 66-40 — Dissolve 28 g of crude Antibiotic 66-40 in 100 ml of distilled water and charge to an anion exchange adsorption column (Dowex 1 X2) in the hydroxyl form. Slurry 2,000 g of the resin in water in to a column 2yj in diameter and 36 "high. Elute the column with distilled water at a rate of about 23 ml/min collecting 100 ml fractions and monitor with a conductivity meter and by disc testing against Staphylococcus aureus. 

Before commencing the elution titrate 10.0 mL of the 0.3 M sodium nitrate with the standard silver nitrate solution, and retain the product of this blank titration for comparing with the colour in the titrations of the eluates. When the titre of the eluate falls almost to zero (i.e. nearly equal to the blank titration) — ca 150 mL of effluent — elute the column with 0.6M sodium nitrate. Titrate as before until no more bromide is detected (titre almost zero). A new blank titration must be made with 10.0 mL of the 0.6M sodium nitrate. 

Dry the organic solvent layer through 80 g of anhydrous sodium sulfate on a glass funnel and collect the dried solution in a 300-mL round-bottom flask. Evaporate the solvent under reduced pressure. Dissolve the residue in 150 mL of n-hexane and transfer the solution into a 300-mL separatory funnel. Extract twice with 100 mL of acetonitrile. Combine the acetonitrile extracts in a 500-mL round-bottom flask and evaporate the solvent under reduced pressure. Dissolve the residue in a small amount of column-eluting solvent (dichloromethane-n-hexane, 1 1, v/v) and transfer the solution to the top of the silica gel column. After eluting the column with 60 mL of solvent of the same composition (discard), elute orbencarb and I with 150mL of dichloromethane. Collect the eluate in a 300-mL flask and evaporate the solvent under reduced pressure. Dissolve the residue in an appropriate volume of acetone for analysis. 

To the flasks for the crop and soil samples (Section 6.1), add 2mL of 0.01 M Tris-HCl buffer solution (pH 7.7) and 50 and 100 qL of 1M Tris-HCl buffer solution for wheat grain, bariey grain and rice straw, and for soil, respectively. Adjust the pH to about 7.7 (confirm the pH with a pH test paper using the sample of untreated area). Homogenize the residue with ultrasonication and transfer the homogenate to the top of an ion-exchange column. Wash the flask twice with 2mL of 0.01 M Tris-HCl buffer solution and transfer the washings to the column. Elute the column with 40 mL of the same buffer solution. Discard this eluate. 

Quantitatively transfer the hydrolysis reaction solution to a 50-mL glass culture tube with a screw-cap by rinsing witli 3x5 mL of deionized water followed by 5 mL of 30% (v/v) sulfuric acid and one additional 5 mL of deionized water. Rinse the Teflon culture tube with acetone and transfer to the glass culture tube. Extract the acidic aqueous phase (pH 1) with 3 x 2.5 mL of toluene. Pass each upper toluene phase through approximately 3 g of anhydrous sodium sulfate contained in a 6-mL disposable filtration cartridge into a 10-mL volumetric flask. Adjust the volume of the solution to 10 mL with toluene. Condition a 3-mL diolsilane bonded silica gel SPE cartridge with two column volumes of toluene. Load a 5-mL aliquot of toluene solution and collect the eluate in a 125-mL round-bottom flask. Elute the column with an additional 50 mL of toluene (use the 75-mL reservoirs) and collect the eluate in the same round-bottom flask. Concentrate the toluene extract to approximately 3.0 mL at 40 °C under weak reduced pressure with a rotary evaporator. 

Prepare a silica gel column as mentioned in Section 3. Transfer the solution derived from Section 6.1.1 to the column. Wash the flask with a small volume of n-hexane-ethyl acetate (9 1, v/v) mixture and elute with the same solvent mixture. Discard the first 50 mL of eluate and collect the next 110 mL of eluate in a 200-mL round-bottom flask (mepanipyrim fraction). Then, elute the column with 100 mL of n-hexane-ethyl acetate (1 1, v/v) mixture and collect the eluate (propanol form the metabolite fraction). Evaporate the solvents under reduced pressure and dissolve the residue in an appropriate volume of acetone for analysis. 

After the glauclne had been eluted, the column was washed with 50% methanol in ether. All fractions were assayed, and only the fraction containing dehydrogalucine was active. Dehydroglaucine was subsequently assayed and was shown to be the antimicrobial agent present in fractions 12-19 (Table II). 

For one stream, the loading solution (2mM acetic acid) initially flowed at 4.0 mL/min through the extraction column to waste for approximately 1 min. The column switching valve then switched so that the mobile phase eluted analytes from the extraction column onto the analytical column. During the elution step, the loading pump transmitted the wash solution directly to waste. After elution, the column switching valve returned to its original position to wash (weak base solution followed by 100% MeOH) and re-equilibrate (2mM acetic acid) the extraction column with a... 

Elute the column with 75ml of acetone and collect the eluate in a 100ml round-bottomed flask this fraction contains the Oxamyl. Using the same conditions as above, evaporate the eluate just to dryness in a rotary evaporator and quantitatively transfer the residue into a 1.0 or 2.0ml calibrated flask with small portions of ethyl acetate. Make up to volume with ethyl acetate and shake thoroughly. 

Most process scale chromatographic separations are run under isocratic conditions, and therefore robust solvent recycling processes need to be designed. Figure 12.16 presents a simplified scheme coupling chromatography with eluent recycling [11]. The solvent is recovered from both evaporators and dryers while pure dry compounds are recovered. The recycled solvent is reused to elute the column and to dissolve the dry feed mixture. Only a small amount of fresh solvent is automatically added to the recycled solvent in order to adjust the eluent composition. 

Six 25-mL portions of distilled ethyl ether. The first 25-mL portion of distilled ether was added to the resin column, and the column was agitated to free the resin. The column was then allowed to stand for 5-10 min before draining. The second through sixth 25-mL portions of ether eluants were used for desorption by gravity flow without agitation and were combined with the first aliquot. In some experiments, additional 25-mL aliquots of ether were used to elute the column. 

The procedure for gel column chromatography is very similar to the general description given earlier. The same precautions must be considered in packing, loading, and eluting the column. A brief outline of important considerations follows. 

For purification, load the reaction mixture onto a 9 x 200 mm Bio-Gel P-6DG column packed in PBS (0.1% BSA may be added as a carrier to the PBS to reduce loss of streptavidin by adsorption to the column) Elute the column with PBS and collect 0.5-mL fractions The first set of radioactive fractions (as determined by counting in a y-ray counter) contains radioiodinated streptavidin, while the unreacted radioiodine elutes in the later fractions. Pool the radioiodinated streptavidin fractions. 

The checkers eluted the columns with a slight positive air pressure on the solvent reservoir to prevent formation of gas bubbles and cracks in the chromatographic medium. Fractions were collected in 25-mL test tubes (Note 1), analyzed by TLC on silica gel, eluting with the column solvent, and visualized with a phosphomolybdic acid solution. The checkers observed a nonvolatile hydrocarbon material (not substrate related) which was eluted in the fractions just prior to the products, which are quite nonpolar themselves and are eluted in the early fractions, ahead of any unreacted ester. Colored, metal-containing components usually remain near the top of the column, although some colored material may accompany the... 

Under vacuum, pre-elute the column with the least polar combination of the required solvents in which the product mixture is readily soluble. If possible, use a single least-polar component (e.g. use pentane for pentane/ether gradient elution). If the silica has been packed correctly, the solvent front will be seen descending in a horizontal line. If channelling occurs, suck the column dry and repeat the packing procedure. Keep the surface of the silica covered with solvent... 

Eluting the column isocratically with hexane/tetrahydrofuran (90.25/9.75, v/v) provides separation of the above overlapped peaks (11). 

Finally elute the column with five-column volumes of 0.05 M Tris-HCl, 1 M NaCl, pH 8.5 This should remove any remaining proteins and allow the column to be re-equilibrated with 0.05 M Tris-HCl, pH 8.5, for re-use or washed with 0.02 (w/v) sodium azide for storage if reuse is not imminent. 

In methods involving a gradient elution, the column geometry has more of an influence and it is preferable to have a length to diameter ratio of at least 5. 

After elution, the columns are regenerated with regeneration buffer, followed by distilled water and equilibration buffer, and stored at 0-4°C in 20% v/v ethanol. Affinity chromatography is also utilized to test the capability of a lead ligand to purify the target protein from crude extracts (see Note 10 and Fig. 5). 

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