Preparing a column

The preparation of an ion-exchange column consists of filling and, if necessary, conversion to another ionic form. It is assumed here that the column is a burette. 

In a typical resin, the diameter of the biggest particles is at least twice that of the smallest, so that the biggest have at least eight times the volume of the smallest. The two main requirements of the filling operation are that these very unevenly sized particles must be packed reasonably homogeneously throughout the full depth of the column, and that no air bubbles must be admitted. 

Half-fill a measuring cylinder with water and transfer a slight excess (say 10%) of resin into it, using a spatula. 

Pour it into a beaker and add an excess of the appropriate acid or base, e.g. hydrochloric acid for a tertiary amine resin or sodium hydroxide for a sulphonic acid. It is convenient but not essential to use approximately molar reagents, in aqueous solution. 

Stir for a few minutes neutralisation is rapid, but the neutralising reagent needs a certain amount of time to penetrate to the innermost regions of the resin matrix. 

---

Conversion of the salt of a weak base into the free base. Prepare a column of a strong base anion resin (such as Amberlite IRA-40o(OH) ) washed with distilled water as above. Drain off most of the water and then allow 100 ml. of A//2.Na.2C03 solution to pass through the column at 5 ml. per minute. Again wash the column with 200 ml. of distilled water. Dissolve 0-05 g. of aniline hydrochloride in 100 ml. of distilled water and pass the solution down the column. The effluent contains aniline in solution and free from all other ions. 

Removal of acids from mixtures of acids and neutral substances. Prepare a column of a strong base anion resin and treat it with sodium... 

Procedure. Prepare a column of the anion exchange resin using about 15 g of Duolite Al 13 in the chloride form (Section 7.9). The column should be made up in 2 M hydrochloric acid. 

Prepare a column of Sepharose-4B (Pharmacia) in 2mM-Tris-HC1. pH 7.6, 0.1 mM-EDTA in a 1 ml disposable plastic pipette... 

Prepare a column of G-75 or G-100 Sephadex (Fine) in a buffer containing 0.75 M NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM-EDTA in a disposable 1 ml plastic pipette as before. Apply the sample and chromatograph collecting two drop fractions. 

Prepare a column for chromatography with silica gel slurried in dichloromethane, so that the column height is c. 30 cm. Load the solution... 

Prepare a column for chromatography using neutral alumina (activity II—III,... 

Remove the hexane on a rotary evaporator to yield crude 2-bromophosphinine. Prepare a column for flash chromatography using silica gel and hexane as solvent, and chromatograph the residue rapidly under a slight nitrogen pressure. 

Approximately 40 mg of hemoglobin is applied to a 0.9 X 45 cm column of DEAE-Sephadex and the chromatogram is developed with a gradient of 0.05 ilf Tris-HCl buffers (D8). Fractions containing Hb-F are pooled, the pooled effluent deluted once with water, and one or two drops of a 2% KCN solution are added. The pH of this solution is adjusted to 6.5-6.7 with 1.0 M maleic acid. CM-Sephadex (C-50, capacity 4.5 0.5 mEq/g, particle size 40-120 p, Pharmacia Fine Chemicals) which is equilibrated with 0.05 M Tris-maleic acid buffer, pH 6.5, is used to prepare a column of 0.5 X 2.0 cm for concentrating the hemo-... 

La. The material is hardly affected by hot aqueous solutions and is particularly effective in absorbing corrosion-product ions derived from metal surfaces. Kraus, Carlson and Johnson (1956) prepared a column from precipitated zirconium tungstate for the separation of Na+, K+, Rb+ and Cs+. The eluant was aqueous NH4CI whose concentration was increased as the elution proceeded. 

With total reflux no product is obtained. This is an unsatisfactory condition for all parties concerned. What is desired is to be able to determine prior to distillation just how many plates are needed and to prepare a column accordingly. Too many plates waste energy and time, and too few plates produce an inferior product. The equations developed below show how the number of plates required for any system can be determined. Refer to Figure 3-13. Assume... 

For multiple-compound separations, two techniques are common. The first, which is more common, is to prepare a column for each compound desired and stack the columns vertically on a tall ring stand (Figure 15-11). The second method is to use a larger column and place the packings on top of each other. About 2 mm of dry Celite is packed firmly between each section, so they can be separated easily. 

Sample preparation Prepare a column as follows. Swirl Chelating Sepharose Fast Flow resin (Pharmacia) in its bottle, add it to a potypropylene column to give a bed volume of... 

After overnight incubation at room temperature, remove excess free glutaraldehyde by gel filtration. To do this, use a gel matrix with an exclusion limit of 20,000"C50,000 for globular proteins. Use medium-sized beads (approx 100 pm in diameter). Prepare a column with 5 mL of bead volume according to the manufacturer s instructions. To make the column easier to load and run, first add 20 pL of glycerol and 20 pL of 1% xylene cylanol. The column should be prerun with a minimum of 10 column vol of 0.15 MNaCl. Allow the column to run until the buffer level drops just below the top of the bed resin. Stop the flow of the column. Carefully load the column with the glutaraldehyde-treated HRP. Release the flow and allow the HRP to run into the column. Just as the level of the HRP solution drops below the top of the column, carefully add 0.15 MNaCl. Run the column with 0.15 MNaCl. Pool the fractions that look brown. These contain the active enzyme. 

Sample preparation Prepare a column as follows. Swirl Chelating Sepharose Fast Flow resin (Pharmacia) in its bottle, add it to a polypropylene column to give a bed volume of 1.0-1.2 mL, wash 3 times with 2 mL portions of water, wash with 2 mL 10 mM copper sulfate, wash with two 2 mL portions of water. Centrifiige 5 mL milk at 10° at 1500 g for 15 min, remove the lower layer and add it to 10 mL succinate buffer, mix, centrifuge at 1500 g for 30 min, add the supernatant to the column. Wash with 2 mL succinate buffer, wash with 2 mL water, wash with 2 mL MeOH, wash with 2 mL water, wash with 700 pL citrate/phosphate buffer (be careful not to disturb bed), elute with 2.5 mL cit-rate/phosphate buffer (column is white and eluate is blue). Filter (Amicon Centricon 30,... 

Prepare a column containing about 40meq of a strongly basic anion exchanger chloride in 80% methanol. 

Prepare a column containing 10 milliequivalents of a strongly acidic cation-exchange resin in the acid form, in 80% ethanol. Wash with 100 ml 80% ethanol. 

Prepare a column with an equal mixture of sodium carbonate and Silflow. Pack the column dry and tamp down before use. 

---