Preparative-scale chromatography column preparation

As with microscale columns, the procedures described in this section should be followed carefully in preparing a semimicroscale or conventional-scale chromatography column. Failure to pay close attention to the details of these procedures may adversely affect the quality of the separation. 

Detection requirements in preparative-scale chromatography also differ from analytical erations where detectors are selected for their sensitivity. Sensitivity is not of overriding importance in preparative-scale chromatography the ability to accommodate large column flow rates and a wide linear response range are more useful. The sensitivity of the refractive index detector is usually quite adequate for prqtaratlve work but the ... 

So far we have considered preparative-scale chromatography when the column is not overloaded and the desired amount of ,sample... 

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. 

Figure 11. Schematic of the inlet end piece in cross section of the preparative scale porous silica chromatography column. All parts are Teflon unless otherwise noted. The thermostat jacket is not shown...

Scaling factors between typical column dimensions for preparative and large-scale chromatography are summarized in Appendix Al. 

Radial compression uses radial pressure applied to a flexible-wall column to lessen wall effects. The mobile phase has a tendency to flow slightly faster near the wall of the column because of decreased permeability. The solute molecules that happen to be near the wall are carried along faster than the average of the solute band, and, consequently, band spreading results. Preparative scale radial compression chromatography columns have been found to possess efficiencies close to those of analytical-scale columns when an adequate radial compression level is used. Radial compression technology also helps lower the cost by substituting reusable column holders in place of expensive steel columns. 

However, these pumps tend to be expensive. With such pumps, direct pumping recycle chromatography is impossible. Another disadvantage is the limited volume of solvent held in the cylinder (although sometimes two pumps can be used in tandem). This volume is 250-500 ml for most of these pumps, whereas the flow--rate through LC columns is usually 0,2 to a few ml/min, so that this disadvantage is not serious in analytical separations, but remains important in preparative--scale chromatography. 

In elution chromatography the mobile and stationary phase are normally at equilibrium. The sample is applied to the column as a discrete band and sample components are successively eluted from the column diluted by mobile phase. The mobile phase must compete with the stationary phase for the sample components and for a separation to occur the distribution constants for the sample components resulting from the competition must be different. Elution chromatography is the most convenient method for analysis and is commonly used in preparative-scale chromatography. 

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