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Therefore, typical sample matrices that can be employed in normal-phase SPE include hydrocarbon or fatty oils diluted in an organic solvent, hexane, isooctane, chlorinated solvents, THF, diethyl ether, and ethyl acetate. Most organic analytes exhibit some polar functionalities that can be exploited for normal-phase separation. Because many molecules exhibit polar functionality, each interaction can provide different levels of selectivity offering highly selective separations of compounds very similar in structure. Normal-Phase SPE __Retention Mechanism__ Polar Interactions - Hydrogen bonding, π-π, dipole-dipole, and induced dipole-dipole __Sample Matrix__ Non-polar samples - Organic extracts of solids - Very non-polar solvents - Fatty oils, hydrocarbons __Analyte Characteristics__ Analytes exhibiting polar functionalities - Hydroxyl groups, carbonyls, amines, double bonds - Hetero atoms (O, N, S, P) - Functional groups with resonance properties __Elution Scheme__ Polar interactions disrupted with a more polar solvent or solution - Acetonitrile, methanol, isopropanol - Combinations of buffer/solvent or solvent/solvent mixtures __Common Applications__ - Cleanup of organic extracts of soils and sludge - Fractionation of petroleum hydrocarbons - PCBs in transformer oil - Isolation of compounds in cosmetics ## [](https://www.sigmaaldrich.com)Basic Steps 1. __Sample Pre-treatment__ Liquid samples should be initially extracted or diluted with a non-polar solvent such as hexane or a chlorinated solvent. Soil, sediment, and other solid samples are initially extracted (Soxhlet or sonication) with a non-polar solvent, and concentrated prior to SPE cleanup. Aqueous residues in the sample can reduce normal-phase retention. It may be necessary to further dry the organic extract with sodium sulfate or magnesium sulfate prior to SPE. 2. __Conditioning/Equilibration__ Condition and equilibriate with 2-3 tube volumes of a non-polar solvent similar or identical to sample matrix resulting from sample pre-treatment. 3. __Sample Load__ Apply sample (from step 1) at a consistent and reduced flow rate of ~1-2 drops/second to ensure optimal retention. The compounds should be a non-polar solvent (e.g., hexane) for optimal retention. Note that methanol and acetonitrile are often used as elution solvents in normal-phase SPE, and will often not promote compound retention during sample load. 4. __Wash__ Sample interferences are often co-retained with compounds of interest during sample load. A wash step is necessary to elute interferences without prematurely eluting compounds of interest. In normal-phase SPE, 1-2 tube volumes of the solvent used in sample pre-treatment and conditioning can be used during wash. 5. __Elution__ Disrupt polar interactions with a solvent or solvent/buffer mixture more polar than both the sample and wash solutions. Typical elution solvents include water miscible organic solvents such as acetone, acetonitrile, methanol, and isopropanol. Eluting with increasingly polar solvents (or solvent mixtures) in succession can also fractionate multiple compound classes. See “Common Normal-Phase Solvents” table for assistance. 6. __Eluate Post-treatment__ Normal-phase SPE is often followed by GC analysis, and therefore requires a volatile sample matrix prior to injection. Use sodium sulfate or magnesium sample to remove residual moisture. Further SPE eluate concentration may also be necessary prior to analysis.[](https://www.sigmaaldrich.com) ### Common Normal-Phase Solvents __Solvent____Elutropic (e°) or__ __elution strength__ __on silica____Promotes__ __Normal-Phase__ __Retention / Elution__ Hexane0.00__Retention__ Isooctane0.00 Carbon tetrachloride0.14 Toluene0.22 Benzene0.27 Tert-butyl methyl ether0.29 Chloroform0.31 Methylene chloride (dichloromethane)0.32 Diethyl ether0.29 Ethyl acetate0.43 Tetrahydrofuran0.35 Acetone0.45 Acetonitrile0.50 40% methanol in acetonitrile0.67 20% methanol in diethyl ether0.65 20% methanol in methylene chloride0.63 Isopropanol0.63 Methanol0.73 Water>0.73__Elution__ Acetic acid>0.73 ## Materials 申し訳ございませんが、想定外のエラーが発生しました。 Response not successful: Received status code 500 __Related Articles__ - [Empowering Biochemists to Analyze Glycoproteins with Exceptional Reproducibility](https://www.sigmaaldrich.com/JP/en/technical-documents/protocol/analytical-chemistry/large-molecule-hplc/bioshell-glycan-hplc-columns) - 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