Combination with Solid-Phase Extractors

Solid-phase extractors (SPEs) can be used separately from the HPLC instrumentation to purify and concentrate the sample, but can also be filled in small precolumns and coimected with the injector for a higher degree of automation. 

A commonly used operation is determination of hydrophobic drugs in biofluids with restricted access materials (RAMs). On such SPEs, the small hydrophobic components enter the pores and are retained in the hydrophobic stationary phase material in the pores, while proteins and hydrophilic compounds are washed out of the precolumn. Finally, the hydrophobic solutes are eluted from the pores with methanol/acetonitrile. 

The use of RAMs in biological fluids is described in more detail in Ref [2]. 

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For the purposes of this method, a soil/sediment sample is defined as a portion of wet soil/sediment which does not contain oil, but which may contain other solids such as stones, vegetation, etc. The sample should not contain an obvious liquid phase (see Section 8.4). A 10 g aliquot of the soil/sediment sample is spiked with the internal standard solution and extracted with toluene in a combination of a Soxhlet extractor and a Dean Stark water separator (SDS). 

B. Tropohne. In a 1-1., three-necked, round-bottomed flask equipped with a mechanical stirrer, addition funnel, and reflux condenser are placed 500 ml. of glacial acetic acid and then, cautiously, 100 g. of sodium hydroxide pellets. After the pellets have dissolved, 100 g. of 7,7-dichlorobicyclo[3.2.0]hept-2-en-6-one is added and the solution is maintained at reflux under nitrogen for 8 hours. Concentrated hydrochloric acid is then added until the mixture is about pH 1 approximately 125 ml. of acid is required. After the addition of 1 1. of benzene, the mixture is filtered and the solid sodium chloride is washed with three 100-ml. portions of benzene. The two phases of the filtrate are separated and the aqueous phase is transferred to a 1-1. continuous extractor (Note 8) which is stirred magnetically. The combined benzene phase is transferred to a 2-1. pot connected to the extractor and the aqueous phase is extracted for 13 hours. Following distillation of the benzene, the remaining orange liquid is distilled under reduced pressure... 

Oxazine-2-one (3). A suspension of 2 (51 g, 239 mmol) in THF (1200 mL) was treated with ethyl bromoacetate (60 g, 359 mmol) and TEA (49 g, 485 mmol). After 18 h stirring, TEA.HBr was filtered and the filtrate evaporated in vacuum. The residue was washed with water and recrystallized from EtOH (250 mL) to yield 60.3 g (84%) of products. To 239 g (80 mmol) of this product in 160 mL of CHCls was added a mixture of di-tert-butyl dicarbonate (34.9 g, 160 mmol), NaCI (32.8 g, 560 mmol) and saturated solution of NaHCOs (160 mL) and the mixture was refluxed for 20 h. The aqueous phase was extracted with CHCls, and the combined organic layers washed (water), dried (Na2S04), the solvent evaporated and the residue distilled to remove the di-tert-butyl carbonate. The crude product was used for the next step. Recrystallized (hexane EtOAc 3 1), mp 60-62 C, = -20.5 (c 5.5 CH2CI2). To the crude product above (32 g), in PhH (750 mL) was added pTsOH (1.5 g, 8 mmol), the mixture was refluxed (Soxhiet extractor filled with CaCb for 8 h, the solid after removal of solvent was dissolved in CH2CI2, washed (water) and evaporated. Recrystallization from EtOH (750 mL) gave 20.7 g of 3 (73%). 

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