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Questions and Answers
What ions are replaced when hard water is treated with a cation-exchange resin in Na+ form?
What ions are replaced when hard water is treated with a cation-exchange resin in Na+ form?
Ion-pair chromatography is primarily used for separating uncharged analytes.
Ion-pair chromatography is primarily used for separating uncharged analytes.
False
What does SEC stand for in chromatography?
What does SEC stand for in chromatography?
Size Exclusion Chromatography
In an anion-exchange resin, the mobile phase typically contains _____ ions that replace anions in the water.
In an anion-exchange resin, the mobile phase typically contains _____ ions that replace anions in the water.
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Match the chromatography techniques with their primary applications:
Match the chromatography techniques with their primary applications:
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What happens to Cu(NO3)2 when passed through an anion-exchange and cation-exchange resin?
What happens to Cu(NO3)2 when passed through an anion-exchange and cation-exchange resin?
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In size exclusion chromatography, large molecules are retained the longest.
In size exclusion chromatography, large molecules are retained the longest.
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What is the purpose of adding a surfactant in ion-pair chromatography?
What is the purpose of adding a surfactant in ion-pair chromatography?
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What kind of molecules are predominantly separated using Ion-Exchange Chromatography?
What kind of molecules are predominantly separated using Ion-Exchange Chromatography?
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In Ion-Exchange Chromatography, cations can only exchange with cations.
In Ion-Exchange Chromatography, cations can only exchange with cations.
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Name two types of stationary phases used in Ion-Exchange Chromatography.
Name two types of stationary phases used in Ion-Exchange Chromatography.
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In Size Exclusion Chromatography, as the size of analytes decreases, the elution volume __________.
In Size Exclusion Chromatography, as the size of analytes decreases, the elution volume __________.
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Match the following chromatographic techniques with their characteristics:
Match the following chromatographic techniques with their characteristics:
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Which of the following factors increases affinity in Ion-Exchange Chromatography?
Which of the following factors increases affinity in Ion-Exchange Chromatography?
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In Size Exclusion Chromatography, larger molecules elute before smaller molecules.
In Size Exclusion Chromatography, larger molecules elute before smaller molecules.
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What type of polymer is commonly used to prepare gels in Ion-Exchange Chromatography?
What type of polymer is commonly used to prepare gels in Ion-Exchange Chromatography?
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What is a disadvantage of lightly cross-linked resins in ion-exchange chromatography?
What is a disadvantage of lightly cross-linked resins in ion-exchange chromatography?
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Strong ion exchangers lose their ionization at some pH values.
Strong ion exchangers lose their ionization at some pH values.
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What type of molecules are various ion-exchange resins well suited for?
What type of molecules are various ion-exchange resins well suited for?
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Tertiary anion exchangers lose their ability to bind anions in ___________ solutions.
Tertiary anion exchangers lose their ability to bind anions in ___________ solutions.
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Match the types of ion exchangers with their characteristics:
Match the types of ion exchangers with their characteristics:
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Which of the following describes gradient elution in ion exchange chromatography?
Which of the following describes gradient elution in ion exchange chromatography?
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In anion exchange chromatography, increasing the eluent anion concentration enhances the analyte's interaction with the resin.
In anion exchange chromatography, increasing the eluent anion concentration enhances the analyte's interaction with the resin.
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What effect does swelling have on lightly cross-linked resin selectivity?
What effect does swelling have on lightly cross-linked resin selectivity?
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Study Notes
Instrumental Analysis I - Lecture 8-WS24: Chromatographic Mechanisms for HPLC Applications
- HPLC applications use chromatographic mechanisms for separation.
- Chromatographic mechanisms depend on the stationary phase type.
- Key types of separations include Adsorption, Partition, Ion Exchange, Ion Pair, and Size Exclusion.
Ion Exchange Chromatography
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Common for separating charged biological molecules (proteins, peptides, amino acids, nucleotides).
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Ion exchange uses resins or gels with charged functional groups.
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Ions with higher affinity displace those with lower affinity.
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Exchange mechanisms depend on:
- Affinity increases
- Size of solvated ions decreases
- Polarizability increases
Stationary Phases in Ion-Exchange Chromatography
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Resins are amorphous organic materials.
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Gels are amorphous organic materials that are softer.
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Cellulose and dextran gels: Dextran cross-linked to glycerin.
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Polystyrene resins: Made by co-polymerization of styrene and vinyl-bearing molecules.
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Importance of cross-linked resins:
- Advantage: Rapid equilibration of solutes
- Disadvantage: Swelling in water decreases the density of ion exchange sites leading to reduced selectivity.
Strong vs. Weak Ion Exchangers
- Strong exchangers: Remain ionized at all pH values. Example: SO3.
- Weak exchangers: Lose ionization at certain pH values. Example: CO2.
Examples of Ion-Exchange Resins
- Different types (strongly/weakly acidic, cation/anion) have different chemical compositions and usual forms.
- Resin types have varying thermal stability and selectivity.
Gradient Elution in Ion-Exchange Chromatography
- Elution uses concentration gradients of anions in eluents.
- Gradient techniques increase pH, increasing the concentration of the eluent anion.
- Used to displace components at different rates based on their affinities.
Ion-Exchange Chromatography for Water Treatment
- Softening hard water: Replacing Ca²⁺ with Na⁺. This also applies if CaSO₄ is present.
- Deionizing water: Resin exchange with H⁺ and OH⁻ removes charged molecules. This applies if Cu(NO₃)₂ is present.
Ion-Pair Chromatography
- Used for separating charged analytes, especially acids and bases.
- Stationary phase has a surfactant that attracts anions.
- Separates analyte cations through electrostatic attractions to the surfactant.
Size Exclusion Chromatography (SEC)
- Separation of mixtures based on molecular size.
- Smaller molecules enter the pores of the stationary phase more readily.
- Larger molecules elute faster as they do not enter the pores.
- Methods include Gel Filtration Chromatography (GFC) and Gel Permeation Chromatography (GPC).
Chromatographic Parameters in SEC
- Vt: Column total volume.
- Vg: Volume of solid matrix.
- Vi: Volume outside the solid matrix.
- Ve: Elution volume, directly proportional to retention time.
Determining Molecular Weights by SEC
- Calibration graphs establish the relationship between elution volume and molecular weight.
- Molecular weight determination in SEC requires a precise calibration graph.
Flow Charts for Small/Large Molecules
- Charts guide selection of chromatographic separation methods based on molecular weight and solubility characteristics.
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Description
Explore the chromatographic mechanisms used in HPLC applications in this quiz. Focus on ion exchange chromatography, its applications, and the influence of stationary phases. Test your knowledge on various separation techniques critical in instrumental analysis.
