Questions and Answers
What is the main purpose of Reversed Phase SPE?
Which type of sorbent is typically used in Ion Exchange SPE?
What is the first step in Conditioning the cartridge for SPE?
Which type of interactions primarily lead to the retention of an analyte in Normal Phase SPE?
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In Anion Exchange SPE, what types of molecules are retained by the sorbent?
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What is the final step in the Elution protocol in Solid Phase Extraction?
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What is the purpose of column conditioning in Solid Phase Extraction (SPE)?
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In Solid Phase Extraction (SPE), what is the purpose of sample loading?
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What is done during the step of washing the packing in Solid Phase Extraction (SPE)?
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In Solid Phase Extraction (SPE), what is done during the elution step?
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Which type of Solid Phase Extraction (SPE) uses a solution that removes compounds of interest but leaves impurities in the packing?
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What is an essential factor to consider in the elution protocol for Solid Phase Extraction (SPE)?
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What is the general elution protocol for REVERSED PHASE SPE?
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Which type of sorbent is commonly used in REVERSED PHASE SPE?
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What is the purpose of conditioning the cartridge in SPE?
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Which type of SPE can be used for compounds that are charged when in solution?
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In REVERSED PHASE SPE, how should the more tightly bound components of interest be eluted?
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What type of compounds can be suitable for Anion Exchange SPE?
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Study Notes
Solid Phase Extraction (SPE)
- Analyte molecules are enriched on the adsorbent, while interfering components and solvent molecules (matrix) are not retained
- Remaining interfering components are washed from the adsorbent, and the analyte is removed from the adsorbent by elution with a suitable solvent
Retention of Interfering Components
- Analyte molecules show no interaction with the adsorbent, whereas interfering components and solvent molecules (matrix) are retained
- Analyte molecules are "washed" from the adsorbent, and the solid phase is used to "filter" the sample
Normal Phase SPE
- Involves a polar analyte, a mid- to nonpolar matrix, and a polar stationary phase
- Typically uses sorbents such as polar-functionalized bonded silicas (e.g. LC-CN, LCNH2, and LC-Diol) and polar adsorption media (e.g. LCSi, LC-Florisil, ENVI-Florisil, and LC-Alumina)
- Retention of an analyte is due to interactions between polar functional groups of the analyte and polar groups on the sorbent surface, including hydrogen bonding, pi-pi interactions, dipole-dipole interactions, and dipole-induced dipole interactions
- Conditioning the cartridge with a nonpolar solvent, usually the sample solvent, is required
Steps in SPE
- Column conditioning: to wash the extraction system and remove potential interferents, and to activate the solid sorbent
- Sample loading: sample is forced through the sorbent material, and the analyte of interest is retained by the sorbent
- Washing the packing: unwanted materials are washed off using the same solution
- Eluting the compound of interest: rinsing the packing with a small volume of a solution that removes compounds of interest, but leaves behind impurities
Normal Phase Elution Protocol
- Elute unwanted components with a nonpolar solvent
- Elute the first component with a polar solvent
- Elute remaining components of interest with progressively more polar solvents
Reversed Phase SPE
- Involves a mid- to nonpolar analyte, a polar (usually aqueous) or moderately polar sample matrix, and a nonpolar stationary phase
- Typically uses sorbents such as alkyl- or aryl-bonded silicas (e.g. LC-18, ENVi-18, LC8, ENVI-8, LC-4, and LC-Ph)
- Retention of organic analytes from polar solutions is due to attractive forces between the carbon-hydrogen bonds in the analyte and the functional groups on the silica surface (Van der Waals forces or dispersion forces)
- Conditioning the cartridge with methanol or acetonitrile followed by deionized water or buffer is required, and the cartridge should not be allowed to dry
- Loading the sample (dissolved in a strongly polar solvent) into the cartridge is required
Reversed Phase Elution Protocol
- Elute unwanted components with a strongly polar solvent
- Elute weakly held components of interest with a less polar solvent
- Elute more tightly bound components of interest with progressively more non-polar solvents
Ion Exchange SPE
- Used for compounds that are charged when in a solution (usually aqueous, but sometimes organic)
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Description
Learn about the general elution protocols for normal phase and reversed phase chromatography, including the steps to separate components based on polarity. Understand the use of nonpolar and polar solvents in these chromatographic techniques.
