Preparative HPLC in practice

An analytical chromatogram generally provides the starting point for preparative separation, and conditions favourable for separating the sample mixture isocraticaUy with good resolution have to be estabhshed  

The structures of the products were not known, nor was there any certainty that each peak represented one component only. 

A preparative high-performance column packed with 5, 7 or 10 pm material and available from various manufactures is used after the analytical separation optimization stage (columns with coarser particles of around 40 pm are more suitable for easy separations requiring less than 100 theoretical plates). A 10 mm i.d. column is designed for 10-100 mg samples, rising to 21.5 mm (o.d. 1 in) for samples between 100 mg and 1 g. The transfer from analytical to preparative conditions works best if both columns are packed with the same stationary phase. 

Thicker columns require a pump that ensures a high flow rate, an identical linear mobile phase throughput and hence comparative retention times, being dependent on the flow rate, increasing with the square of the column diameter 1 ml min for a 4 mm i.d. column rising to 6.25 mlmin for a 10 mm i.d. column  

The detector chosen must be insensitive. A refractive index detector (with a preparative cell if required) or a UV detector with an optical path length of 0.1-0.5 mm 

As the reaction products are relatively volatile, the mobile phase had to have a low boiling point, otherwise hexane-t rf-butylmethyl ether would have been tried. 

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