Column pouring

The Sephadex column may be prepared for another reaction mixture by eluting an additional 15-20 mL of buffer to be sure all components are washed from the column. If you are finished using the column, pour the gel into a container labeled Used Sephadex G-25 . The gel may be recycled and used again. 

When you are finished with the Sephadex column, pour the gel into a container labeled used Sephadex G-50. The gel may be recycled and used later. 

Obtain a prepacked column and clamp it to a ring stand. If you must prepare your own column of IDA-agarose, use a 1 X 6-8 cm column. Pour in about 2 mL of the IDA-agarose slurry. (Be sure the column outlet is closed.) Allow the gel to settle to a column 1-2 cm high. Protect the surface of the gel by allowing a small circle of filter paper to settle onto the top. Allow most of the solution to pass through the column, close the outlet, and add buffer A to fill the column. 

Clamp a column (approximately 1.5 cm x 40 cm) to a stand, and with the outlet closed run a small volume of PBS into the column. Pour the Sephadex G-50 suspension into the colunm and allow to settle until approximately 1 cm of column length is filled. Open the outlet to allow a flow of PBS, which facilitates column packing. Add more Sephadex slurry to give a packed volume of one-third of the column length, with a reasonable depth of PBS above the packed Sephadex. Close the outlet. 

This is a more elaborate column with extensive glass working on the inside to increase the surface area. The glass bulbs are free to move and move up and down randomly during a distillation. This is a noisy, but very effective, column. Pour afew milliliters of solvent down the top of the column before you start the distillation and the bulbs will move freely. Otherwise, one sometimes sticks and the internal pressure blows the column off of the pot. 

Carefully, pour the Sephadex into the column. Pour it down the sidewalls so you do not get any air bubbles in the column. (Normally the column would be filled with liquid before the Sephadex is added. However, it takes a long time to drain away the excess and that is not comparable with the time allotted for this experiment). (NOTE if an air bubble forms in the column, pour out the contents and try again). 

Place a small wad of cotton wool in the lower tip of the upper column, pour in sufficient light petroleum to fill the middle of the 10-mm diameter tube and add alumina (Type 1) to fill the 5-mm diameter tube. Apply the inert gas pressure to the top of the column, and when the excess of light petroleum has been forced through the column, release the pressure and transfer the light petroleum extract to the column. 

When the adsorbent has been introduced into the tube, the latter is fitted into a filter flask (see Fig. 77, 46, 2) to which a pump is attached the pump is run slowly and the column is again pressed down gently with the wooden pestle. The circumference of the upper surface is gently and uniformly tapped, especially where it is in contact with the glass surface, for about one minute air bubbles and channels are thus avoided when the solution is poured in. Some workers place a loose plug of cotton wool or a circle of filter paper at the top of the column in order to protect the solid from disturbance when the liquid is introduced. 

To obtain pure acetaldehyde, the product must be redistilled. Clean and dry the 200-250 ml. flask first used, immerse it in cold or ice water pour in the crude acetaldehyde rapidly, attach the fractionating column, etc. Immerse the receiver in crushed ice. Heat the flask gently in a water bath and adjust the temperature so that the aldehyde distils slowly and at a uniform temperature. The temperature recorded at the top of the column may depend partly upon the temperature of the laboratory, if this is above 21°. Pure acetaldehyde boils at 21°. 

Ethyl n-butyrate. Use a mixture of 88 g. (92 ml.) of n-butyric acid, 23 g. (29 ml.) of ethanol and 9 g. (5 ml.) of concentrated sulphuric acid. Reflux for 14 hours. Pour into excess of water, wash several times with water, followed by saturated sodium bicarbonate solution until all the acid is removed, and finally with water. Dry with anhydrous magnesium sulphate, and distU. The ethyl n-but3rrate passes over at 119 5-120-5°, Yield 40 g. An improved yield can be obtained by distilhng the reaction mixture through an efficient fractionating column until the temperature rises to 125°, and purifying the crude ester as detailed above under methyl acetate. 

Place 200 g, (250 ml.) of rectified spirit in a 1-litre round-bottomed flask fitted with a reflux condenser. Cool in ice and run in, slowly and with frequent shaking, 200 g. (109 ml.) of concentrated sulphuric acid. Add 83 g. (104 ml.) of -butyl cyanide (Section 111,113) to the mixture and reflux the whole for 10 hours. Allow to cool, pour the reaction mixture into ice water, separate the upper layer of ester and alcohol, and dry over anhydrous magnesium or calcium sulphate. Distil through a fractionating column and collect the ethyl n-valerate at 143-146°. A further amovmt of the pure ester may be obtained by redrying the fraction of low boiling point and redistilling. The yield is 110 g. 

Pour the reaction mixture cautiously into 400 g. of crushed ice and acidify it in the cold by the addition of a solution prepared by adding 55 ml. of concentrated sulphuric acid to 150 ml. of water and then coohng to 0°. Separate the ether layer and extract the aqueous layer twice with 50 ml. portions of ether. Dry the combined ethereal solutions over 50 g. of anhydrous potassium carbonate and distil the filtered solution thror h a Widmer column (Figs. II, 17, 1 and II, 24, 4). Collect separately the fraction boihng up to 103°, and the dimethylethynyl carbinol at 103-107° Discard the high boiling point material. Dry the fraction of low boihng point with anhydrous potassium carbonate and redistil. The total 3 ield is 75 g. 

Cholestenone. Place a mixture of 1 0 g. of purified cholesterol and 0-2 g. of cupric oxide in a test-tube clamped securely at the top, add a fragment of Dry Ice in order to displace the air by carbon dioxide, and insert a plug of cotton wool in the mouth of the tube. Heat in a metal bath at 300-315° for 15 minutes and allow to cool rotate the test-tube occasionally in order to spread the melt on the sides. Warm with a few ml. of benzene and pour the black suspension directly into the top of a previously prepared chromatographic column (1) rinse the test-tube with a little more benzene and pour the rinsings into the column. With the aid of shght suction (> 3-4 cm. of mercury), draw the solution into the alumina column stir the top 0 -5 cm. or so with a stout copper wire to... 

---