Normal Phase vs Reverse Phase Chromatography: Quiz and Flashcards

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Questions and Answers

What is the main purpose of Reversed Phase SPE?

Retention of interfering components on the adsorbent (correct)

Conditioning the cartridge with a nonpolar solvent

Retention of the analyte on the adsorbent

Using a polar stationary phase to interact with polar functional groups

Which type of sorbent is typically used in Ion Exchange SPE?

LC-CN and LCNH2

LC-Florisil and LC-Alumina

Anion Exchange resin (correct)

Polar-functionalized bonded silicas

What is the first step in Conditioning the cartridge for SPE?

Condition the cartridge with nonpolar solvent (correct)

Retention of the analyte on the adsorbent

Wash interfering components from the adsorbent

Load the sample into the cartridge

Which type of interactions primarily lead to the retention of an analyte in Normal Phase SPE?

Hydrogen bonding and dipole-dipole interactions Signup and view all the answers

In Anion Exchange SPE, what types of molecules are retained by the sorbent?

Cationic molecules Signup and view all the answers

What is the final step in the Elution protocol in Solid Phase Extraction?

The analyte is removed from the adsorbent by elution Signup and view all the answers

What is the purpose of column conditioning in Solid Phase Extraction (SPE)?

To activate the solid sorbent and remove potential interferents Signup and view all the answers

In Solid Phase Extraction (SPE), what is the purpose of sample loading?

To force the sample through the sorbent material for retention of the analyte Signup and view all the answers

What is done during the step of washing the packing in Solid Phase Extraction (SPE)?

Wash off unwanted materials using a different solution Signup and view all the answers

In Solid Phase Extraction (SPE), what is done during the elution step?

Rinse the packing with a small volume to retain compounds of interest Signup and view all the answers

Which type of Solid Phase Extraction (SPE) uses a solution that removes compounds of interest but leaves impurities in the packing?

Reversed Phase SPE Signup and view all the answers

What is an essential factor to consider in the elution protocol for Solid Phase Extraction (SPE)?

Selecting a solution that leaves behind compounds of interest Signup and view all the answers

What is the general elution protocol for REVERSED PHASE SPE?

Elute unwanted components with strongly polar solvent. Signup and view all the answers

Which type of sorbent is commonly used in REVERSED PHASE SPE?

Alkyl-bonded silicas Signup and view all the answers

What is the purpose of conditioning the cartridge in SPE?

To prepare the sorbent for sample loading. Signup and view all the answers

Which type of SPE can be used for compounds that are charged when in solution?

Ion exchange SPE Signup and view all the answers

In REVERSED PHASE SPE, how should the more tightly bound components of interest be eluted?

With progressively more nonpolar solvents Signup and view all the answers

What type of compounds can be suitable for Anion Exchange SPE?

Charged compounds Signup and view all the answers

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.

Normal Phase vs Reversed Phase Chromatography

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