[Pular para o conteúdo](https://www.sigmaaldrich.com#main-content) [![Merck](https://www.sigmaaldrich.com/static/logos/purple/merck.svg)](https://www.sigmaaldrich.com/BR/pt) Produtos Carrinho0 BRPT Produtos ProdutosAplicativosServiçosRecursosSuporte [Login / Registrar-se](https://www.sigmaaldrich.com/oidc-sign-in) [Nº do Pedido](https://www.sigmaaldrich.com/BR/pt/order-lookup) [Pedido rápido](https://www.sigmaaldrich.com/BR/pt/quick-order) Carrinho0 [Home](https://www.sigmaaldrich.com/BR/en)[Solid Phase Extraction (SPE)](https://www.sigmaaldrich.com/BR/en/applications/analytical-chemistry/sample-preparation/solid-phase-extraction)Solid Phase Extraction: Reversed-Phase Methodology # Solid Phase Extraction: Reversed-Phase Methodology ## Aqueous Sample Matrix/Mobile Phase Environment Reversed-phase SPE is considered the least selective retention mechanism when compared to normalphase or ion-exchange SPE. In other words, it may be difficult for a reversed-phase method or bonded chemistry to differentiate between molecules that are structurally similar. However, because reversed-phase will retain most molecules with any hydrophobic character, it is very useful for extracting analytes that are very diverse in structure within the same sample. Reversed-Phase SPE Retention Mechanism: Non-polar or hydrophobic interactions - Van der Waals or dispersion forces Sample Matrix: Aqueous samples - Biological fluids (serum, plasma, urine) - Aqueous extracts of tissues - Environmental water samples - Wine, beer and other aqueous samples Analyte Characteristics: Analytes exhibiting non-polar functionalities - Most organic analytes - Alkyl, aromatic, alicyclic functional groups Elution Scheme: Disrupt reversed-phase interaction with solvent or solvent mixtures of adequate non-polar character - Methanol, acetonitrile, dichloromethane - Buffer/solvent mixtures Common Applications: - Drugs and metabolites in biological fluids - Environmental pollutants in water - Aqueous extracts of tissues and solids ## Basic Steps 1. __Sample Pre-treatment__ For interference laden samples (e.g., biological fluids), dilute samples 1:1 with buffer. pH manipulation may be important when dealing with ionizable compounds. A compound’s ionization state can drastically change its retention and elution characteristics on a given SPE sorbent. When an analyte is in its neutral form, it becomes more hydrophobic and retention strengthens under reversed-phase conditions. Adjusting the sample pH to 2 pH units above or below the compound’s pKa (depending on the functional group) will effectively neutralize the compound. When dealing with tissues and other solids, conduct a solid-liquid extraction or homogenization using a buffer. Solvents of non-polar character (including methanol and isopropanol) disrupt interaction between the compound and sorbent functional groups. To avoid clogging, it may be necessary to centrifuge, dilute, and/or pre-filter the sample prior to introducing it to the SPE phase. 2. __Conditioning/Equilibration__ Conditioning wets or activates the bonded phases to ensure consistent interaction between the analyte and the sorbent functional groups. Reversed-phase sorbents are often conditioned with 1-2 tube volumes of a water miscible solvent such as methanol or acetonitrile. Equilibration introduces a solution similar to the sample load in terms of solvent strength and pH in order to maximize retention. 1-2 tube volumes of buffer (used in sample pre-treatment) or water are good choices for reversed-phase equilibration. 3. __Sample Load__ Apply sample (from step 1) at a consistent and reduced flow rate of ~1-2 drops/second to ensure optimal retention. 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. 5-20% methanol in water or sample pre-treatment buffer is typical for wash solvents. 5. __Elution__ Disrupt hydrophobic interactions between the analyte and sorbent functional groups with an organic solvent or solvent combination of sufficient non-polar character. Example elution solvents are 1-2 volumes of methanol or acetonitrile. pH manipulation during elution can often improve recovery when dealing with ionizable compounds. In their ionic form, basic and acidic compounds become more polar, weakening reversedphase interaction, possibly allowing for weaker elution solvents and/or reduced elution volumes. 6. __Eluate Post-treatment__ It is often necessary to evaporate and reconstitute the SPE eluate in mobile phase prior to LC analysis. GC analysis often requires further SPE eluate concentration and/or possible matrix exchange with a more volatile solvent. ## [](https://www.sigmaaldrich.com)SPE Tips 1. Drug-protein binding should be disrupted during sample pre-treatment. Strategies include: - 40 µL 2% disodium EDTA per 100 µL mouse plasma - 40 µL 2% formic acid per 100 µL mouse plasma - Other possible reagents (per 100 µL matrix): 40 µL 2% TCA, 40 µL 2% acetic acid, 40 µL 2% TFA, 40 µL 2% phosphoric acid, or 200 µL MeCN (protein ppt.). 2. If the SPE eluate needs to be evaporated prior to analysis, pass vacuum air through the SPE tube for~10 minutes after elution. This will remove residual moisture that may prolong evaporation. 3. Consistent and slow flow rate (1-2 drops per second) during sample load and elution will improve recovery and reproducibility. 4. Reduce bed weight to minimize elution volume. 5. Increase bed weight to retain more polar compounds. 6. Concern for sorbent over-drying is only critical during methanol conditioning. 7. A pre-conditioning solvent such as dichloromethane (or solvent used for elution) can be used before conditioning to remove any impurities on the SPE tube that can interfere with subsequent analysis. ## Materials Desculpe, ocorreu um erro inesperado Response not successful: Received status code 500 __Related Articles__ - [Determination of Formaldehyde and Acetaldehyde in Air Using DNPH Cartridges and Automated On-Line Desorption Followed by HPLC](https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/environmental-testing-and-industrial-hygiene/air-testing/aldehydes-air-dnph) - [Effect of Container Materials on Milk Off-Flavors](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/analytical-chemistry/gas-chromatography/container-materials) - [Pesticide Residue Analysis in Ginger Powder with QuEChERS](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/analytical-chemistry/quechers/analysis-of-pesticide-residues-in-ginger-powder) - [Analysis of Pesticide Residues in Pistachios Using QuEChERS Extraction and Cleanup with Supel™ QuE Z-Sep+](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/analytical-chemistry/quechers/analysis-of-pesticide-residues-in-pistachios) - [HybridSPE-Phospholipid Technology](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/hybridspe-precipitation-technology) - [Impact of Mobile Phase Additives on LC-MS Sensitivity, Demonstrated using Spice Cannabinoids](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/lcms-spice-cannabinoids) - [Solvent Analysis for 40 Per and Polyfluoroalkyl Substances](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/solvent-analysis-for-40-per-and-polyfluoroalkyl-substances) - [Analysis of Drugs of Abuse in Urine](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/analytical-chemistry/solid-phase-extraction/analysis-of-drugs-of-abuse-in-urine-after-cleanup-with-hlb) - [View More](https://www.sigmaaldrich.com/BR/en/search/facet-search?focus=sitecontent&term=facet-search) __Related Product Categories__ - [Solid Phase Extraction (SPE) and QuEChERS](https://www.sigmaaldrich.com/BR/en/products/analytical-chemistry/analytical-sample-preparation/spe-and-quechers) Topo __Faça login para continuar__ Para continuar lendo, faça login ou crie uma conta. 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