Preparative chromatography column loading

The new chemistry is based on a Sr-90/Y-90 separation using a-hydroxyisobutyric acid (a-HIB) and cation exchange chromatography (5). Once the activities are loaded onto the column, the steps to prepare the column for the a-HIB elution remove several of the possibile contaminants including rubidium and cobalt. Finally, the a-HIB elution also removes a wide range of other elements as well, leaving strontium on the ion exchange column (6). 

Monoclonal antibodies against STR were used for the preparation of an immunoaffinity chromatography column. Milk samples were defatted by centrifugation and diluted with phosphate-buffered saline. After loading onto the column, this was washed with saline, and STR and DIHS were eluted with the glycine-HCl buffer. The column bounded 80.4% and 88.7% of milk samples containing 100 ppb STR and DIHS, respectively (117). 

In addition to requiring significant bulk material, the timeframe to complete the isolation is considerable. If the maximum analytical load for a 4.6 mm x 150 mm column has been determined to be 5 mg, assuming the isolation will be performed using semi-preparative chromatography (20 mm x 300 mm column), approximately 190 mg of sample can be loaded onto the preparative column. For a 0.1% level unknown, this translates to 190 pg of unknown injected onto the preparative column. Therefore, a total of 27 injections are required. If the assay time were estimated to be 1 hr, it would take at least 27 hr to perform the injections needed to obtain 5 mg (once again assuming 100% recovery). This timeframe does not include the time needed for method scale-up development, concentration and... 

Preparative-scale chromatography relies on a compromise between three variables (cf. Figure 1) (i) component resolution (determined by selectivity, efficiency and retention factor), (ii) speed of analysis and (iii) column sample capacity (Pescar, 1971). Any two of the desired goals may be realized only at the expense of the third. If a large amount of sample is required in a short time, resolution must be high. If resolution is insufficient, either the column load is limited or the time required for separation is long. 

Clearly it is not possible to describe every technique, or variation in a particular mode of chromatography, so this chapter will concentrate on the most dominant approaches to optimization of preparative HPLC. The objective here is to maximize the column load and to minimize mobile phase wastage allowing the purification of the largest amount of target in the minimum space. 

As the amount of sample injected is increased (increased load) on a chromatographic column, the surface area of column packing material available for interaction decreases. As a result, the resolution of the peaks will decrease. However, in preparative chromatography this is not bad since resolution must be compromised with the load. The extent of this compromise depends upon the individual chromatographer. 

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 silica gel chromatography column using dichloromethane, and load the column by dissolving the residue in the minimum volume of dichloromethane. Run the column with dichloromethane/methanol (199 1, v/v) and the desired dithiol elutes with an of 0.5 (99 1, CH2CI2-MeOH, v/v). Evaporate the eluates containing this material to yield a colourless oil... 

Prepare a silica gel 60 chromatography column (circa 20 g) using pentane/Et20 (1 1, v/v), and then load the column with the solution of the crude product. Run the column with the same solvent mixture until no more tetronate 12a comes off. Triphenylphosphaneoxide will stay at the top of the column under these conditions. Evaporate the eluate on an oil pump and purify the remaining solid by recrystallization from hot Et20 to obtain yellowish needles of 12a m.p. 61°C, 1.27 g (78%). 

Analytical HPLC separations performed with sample loads of < 1 mg/g of column packing material usually do not alter column effectiveness. Preparative separations involve sample loads > 1 mg/g of stationary phase, and in many cases, the higher loading results in reduced selectivity and efficiency. Separation selectivity is of more importance in preparative chromatography than in analytical separations. Resolution (Rs) is affected by several chromatographic variables demonstrated in the following equation 7... 

The last two sections characterized the elements of a chromatographic system and discuss different separation tasks. This section provides guidelines to develop the chromatographic system for a given separation problem. The classical recommendation for preparative separations performed by elution chromatography is as follows Find a mobile phase with high solubility, a compatible stationary phase and then optimize selectivity, capacity and efficiency followed by a systematic increase in column load. 

---