Instrumental Analysis I - Lecture 8: HPLC Mechanisms
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Questions and Answers

What ions are replaced when hard water is treated with a cation-exchange resin in Na+ form?

  • Mg2+ is replaced with Na+
  • Ca2+ and SO4^2- are both removed
  • SO4^2- is replaced with Na+
  • Ca2+ is replaced with 2 Na+ (correct)

Ion-pair chromatography is primarily used for separating uncharged analytes.

False (B)

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.

<p>OH-</p> Signup and view all the answers

Match the chromatography techniques with their primary applications:

<p>Ion-Exchange Chromatography = Water treatment Ion-Pair Chromatography = Separation of charged acids and bases Size Exclusion Chromatography = Separation based on molecule size Gradient Elution = Enhancing separation efficiency over time</p> Signup and view all the answers

What happens to Cu(NO3)2 when passed through an anion-exchange and cation-exchange resin?

<p>Cu2+ is replaced with 2 H+ and NO3- is replaced with OH- (D)</p> Signup and view all the answers

In size exclusion chromatography, large molecules are retained the longest.

<p>False (B)</p> Signup and view all the answers

What is the purpose of adding a surfactant in ion-pair chromatography?

<p>To create an ion-pair surface for the separation of cations.</p> Signup and view all the answers

What kind of molecules are predominantly separated using Ion-Exchange Chromatography?

<p>Charged biological molecules (B)</p> Signup and view all the answers

In Ion-Exchange Chromatography, cations can only exchange with cations.

<p>True (A)</p> Signup and view all the answers

Name two types of stationary phases used in Ion-Exchange Chromatography.

<p>Resins and gels</p> Signup and view all the answers

In Size Exclusion Chromatography, as the size of analytes decreases, the elution volume __________.

<p>decreases</p> Signup and view all the answers

Match the following chromatographic techniques with their characteristics:

<p>Ion-Exchange Chromatography = Separates ions based on charge Ion-Pair HPLC = Uses ions to enhance the solubility of analytes Size Exclusion Chromatography = Separates molecules by size Gradient Elution = Utilizes a changing solvent composition over time</p> Signup and view all the answers

Which of the following factors increases affinity in Ion-Exchange Chromatography?

<p>Decreasing charge of the ions (B)</p> Signup and view all the answers

In Size Exclusion Chromatography, larger molecules elute before smaller molecules.

<p>True (A)</p> Signup and view all the answers

What type of polymer is commonly used to prepare gels in Ion-Exchange Chromatography?

<p>Cellulose or dextran</p> Signup and view all the answers

What is a disadvantage of lightly cross-linked resins in ion-exchange chromatography?

<p>They swell in water, decreasing ion exchange selectivity. (C)</p> Signup and view all the answers

Strong ion exchangers lose their ionization at some pH values.

<p>False (B)</p> Signup and view all the answers

What type of molecules are various ion-exchange resins well suited for?

<p>Large molecules such as proteins.</p> Signup and view all the answers

Tertiary anion exchangers lose their ability to bind anions in ___________ solutions.

<p>moderately basic</p> Signup and view all the answers

Match the types of ion exchangers with their characteristics:

<p>Strong cation exchanger = Remains ionized at all pH values Weak cation exchanger = Loses its ionization at some pH values Strong anion exchanger = Keeps its charge even at low pH Weak anion exchanger = Can lose ion exchange capacity at lower pH</p> Signup and view all the answers

Which of the following describes gradient elution in ion exchange chromatography?

<p>It combines a high concentration of an anionic compound with an increasing concentration of a strong base. (B)</p> Signup and view all the answers

In anion exchange chromatography, increasing the eluent anion concentration enhances the analyte's interaction with the resin.

<p>False (B)</p> Signup and view all the answers

What effect does swelling have on lightly cross-linked resin selectivity?

<p>Decreases selectivity of the resin.</p> Signup and view all the answers

Flashcards

Lightly cross-linked resins

Resins with large pore sizes and low charge densities, ideal for rapid solute equilibration.

Polystyrene resins

A common type of lightly cross-linked resin.

Ion exchange capacity

The ability of a resin to exchange ions with a solution.

Strong ion exchangers

Remain ionized at all pH values; maintains charge regardless of environment.

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Weak ion exchangers

Lose ionization at certain pH values; affected by pH.

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Gradient elution

Technique in chromatography that increases eluent concentration (e.g.,pH) to separate components.

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Anion exchange

A type of ion exchange chromatography that separates anions.

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Eluent

The mobile phase in chromatography; carries the sample through the stationary phase.

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Ion-Exchange Chromatography

A method of separating ions based on their affinity for a charged resin.

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Cation-exchange resin

A resin that selectively attracts and exchanges positively charged ions.

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Anion-exchange resin

A resin that selectively attracts and exchanges negatively charged ions.

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Ion-pair chromatography

A technique used to separate charged molecules using a surfactant in the mobile phase.

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Size Exclusion Chromatography (SEC)

A technique that separates molecules based on their size.

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Gel Filtration Chromatography (GFC)

SEC applied in aqueous solutions for biomolecules.

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Gel Permeation Chromatography (GPC)

SEC applied in nonaqueous solutions for organic polymers.

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Hard water

Water containing high concentrations of dissolved metal ions, often calcium and magnesium.

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Stationary Phases

Resins or gels that attract and hold the ions of interest in Ion-Exchange.

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Resins

Amorphous organic materials (usually hard and rigid) with ion-exchanger surfaces in chromatography.

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Gels

Amorphous organic materials (usually softer and more porous) with ion-exchanger surfaces in chromatography.

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Cellulose and Dextran Gels

Gels used in ion exchange chromatography, often made from polysaccharides.

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Affinity of Ions

The degree to which an ion is attracted to the stationary phase (resin or gel).

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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

  • Common for separating charged biological molecules (proteins, peptides, amino acids, nucleotides).

  • Ion exchange uses resins or gels with charged functional groups.

  • Ions with higher affinity displace those with lower affinity.

  • Exchange mechanisms depend on:

    • Affinity increases
    • Size of solvated ions decreases
    • Polarizability increases

Stationary Phases in Ion-Exchange Chromatography

  • Resins are amorphous organic materials.

  • Gels are amorphous organic materials that are softer.

  • Cellulose and dextran gels: Dextran cross-linked to glycerin.

  • Polystyrene resins: Made by co-polymerization of styrene and vinyl-bearing molecules.

  • 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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Lecture 8 Chromatography Mechanisms PDF

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.

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