Chromatography Techniques and Principles

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What is the purpose of preparative chromatography?

  • To analyze the chemical structure of a compound
  • To separate components of a mixture for further use (correct)
  • To identify the components of a mixture
  • To measure the relative proportion of analyte in a mixture

What is the primary mechanism of separation in gas-liquid chromatography?

  • Partitioning (correct)
  • Ion exchange
  • Size exclusion
  • Adsorption

What is the main factor to consider when selecting a solvent for chromatography?

  • The viscosity of the solvent
  • The polarity of the solvent (correct)
  • The cost of the solvent
  • The boiling point of the solvent

What is the Rf value in paper chromatography?

<p>The ratio of the distance traveled by the compound to the distance traveled by the mobile phase front (C)</p> Signup and view all the answers

Which of the following is NOT a type of chromatographic technique based on the chromatographic bed?

<p>Gas chromatography (C)</p> Signup and view all the answers

Which of these techniques is NOT classified as a type of chromatography?

<p>Spectroscopy (D)</p> Signup and view all the answers

How are spots produced by paper or thin layer chromatography located?

<p>By using a specific type of light, reagents, or radioactive methods (A)</p> Signup and view all the answers

What is the role of the stationary phase in chromatography?

<p>To interact with the sample components and separate them based on their affinities (A)</p> Signup and view all the answers

What does the RR value in chromatography represent?

<p>The ratio between the distances traveled by a given compound and a reference substance in a chromatogram (C)</p> Signup and view all the answers

What is the term for the ratio of the retention time of the test substance, reference compound, and a mixture to the retention time of an internal standard?

<p>Relative retention time (B)</p> Signup and view all the answers

Which combination of effects is most commonly responsible for separation in chromatography?

<p>Adsorption and partitioning (D)</p> Signup and view all the answers

Which of the following is a special technique in chromatography?

<p>Reversed phase chromatography (A)</p> Signup and view all the answers

What is the role of the eluent in liquid chromatography?

<p>To dissolve the sample and carry it through the column (C)</p> Signup and view all the answers

What is the main purpose of using an internal standard in chromatography?

<p>To compensate for variations in the injection volume and flow rate (C)</p> Signup and view all the answers

In which type of chromatography is the retention time used as a parameter for identification?

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

When is a test compound considered the same as the reference compound?

<p>When they have the same retention time (B)</p> Signup and view all the answers

What is the primary function of size-exclusion chromatography (SEC)?

<p>Separating molecules by size (B)</p> Signup and view all the answers

Which of the following statements accurately describes the relationship between particle size and elution time in SEC?

<p>Larger molecules elute faster because they cannot enter the pores and therefore travel through the column more quickly. (A)</p> Signup and view all the answers

What is the role of the stationary phase in SEC?

<p>It provides a porous structure that allows for size-based separation of molecules. (A)</p> Signup and view all the answers

In SEC, what is the primary factor that dictates the separation of molecules?

<p>The volume of the pores that the molecules can access. (A)</p> Signup and view all the answers

Which of the following is NOT a benefit of using SEC?

<p>Direct and accurate determination of the exact molecular weight of a compound. (B)</p> Signup and view all the answers

Why is it important for the analyte to have minimal interaction with the stationary phase in SEC?

<p>To ensure that separation is based solely on size and not on chemical properties. (A)</p> Signup and view all the answers

What is the primary reason why smaller molecules elute later in SEC?

<p>They are more easily retained within the pores of the stationary phase. (B)</p> Signup and view all the answers

Which of the following is NOT a typical application of SEC?

<p>Identifying the specific amino acid sequence of a protein. (B)</p> Signup and view all the answers

Which of the following statements accurately describes the role of the stationary phase in ion exchange chromatography?

<p>The stationary phase is a solid support that contains charged functional groups that interact with analytes based on their charge. (B)</p> Signup and view all the answers

What is the primary mechanism by which analyte molecules are retained on the ion exchange column?

<p>Ionic interactions between the analyte and the stationary phase (A)</p> Signup and view all the answers

In cation exchange chromatography, which type of analyte is selectively retained by the stationary phase?

<p>Cations (C)</p> Signup and view all the answers

What is the primary purpose of using an eluent in ion exchange chromatography?

<p>To introduce oppositely charged ions that compete with the analytes for binding to the stationary phase (B)</p> Signup and view all the answers

Which type of ion exchange chromatography would be most suitable for separating a mixture of negatively charged proteins?

<p>Anion exchange chromatography (C)</p> Signup and view all the answers

Which of the following is a major advantage of ion exchange chromatography compared to other separation techniques?

<p>Its high selectivity based on a single interaction, leading to improved matrix tolerance (C)</p> Signup and view all the answers

What is a significant limitation of ion exchange chromatography?

<p>Its narrow range of applicability, being limited to ionizable molecules (A)</p> Signup and view all the answers

Which type of molecule would likely not be separated effectively using ion exchange chromatography?

<p>Lipids (C)</p> Signup and view all the answers

Which of the following statements describes the elution behavior of molecules in Size Exclusion Chromatography (SEC)?

<p>Larger molecules elute earlier because they can only access a smaller volume of the stationary phase. (C)</p> Signup and view all the answers

What is the 'void volume' in the context of SEC?

<p>The volume occupied by molecules too large to enter the stationary phase. (A)</p> Signup and view all the answers

Which of the following accurately describes the relationship between the 'exclusion limit' and the 'permeation limit' in SEC?

<p>The exclusion limit is the molecular weight of a molecule that cannot enter any pores of the stationary phase, while the permeation limit is the molecular weight of a molecule that can enter all the pores. (A)</p> Signup and view all the answers

Why is it important that all particles in a sample are loaded simultaneously, or nearly simultaneously, in Size Exclusion Chromatography?

<p>To ensure that particles of the same size elute together, leading to a clear separation based on size. (A)</p> Signup and view all the answers

What is the 'eluate' in the context of Size Exclusion Chromatography?

<p>The solution that is collected at the end of the SEC column. (B)</p> Signup and view all the answers

In cation exchange chromatography, what type of molecules are retained by the stationary phase?

<p>Positively charged molecules (B)</p> Signup and view all the answers

Which of the following techniques allows for the gradual variation of counterion concentration to separate ionized molecules?

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

What is the primary principle behind the separation of molecules in ion exchange chromatography?

<p>Differences in charge of molecules (A)</p> Signup and view all the answers

Which of the following is a type of chromatography that utilizes a stationary phase with immobilized charges?

<p>Ion exchange chromatography (C)</p> Signup and view all the answers

What is the main purpose of using exchangeable counterions in ion exchange chromatography?

<p>To compete with ionizable molecules for binding to the immobilized charges (C)</p> Signup and view all the answers

What is the role of pH in ion exchange chromatography?

<p>To influence the charge of ionizable molecules (A)</p> Signup and view all the answers

How does step elution differ from gradient elution in ion exchange chromatography?

<p>Step elution uses a single step change in counterion concentration, while gradient elution uses a gradual change. (B)</p> Signup and view all the answers

In paper chromatography, how does the polarity of the compound affect its travel distance on the paper?

<p>Non-polar compounds travel further because they interact less strongly with the polar cellulose paper. (D)</p> Signup and view all the answers

Flashcards

Chromatography

A laboratory technique for separating mixtures by moving a mobile phase through a stationary phase.

Mobile Phase

The moving solvent that carries the sample through the chromatographic column.

Stationary Phase

The fixed medium in which the sample interacts during separation in chromatography.

Rf Value

The ratio of distance traveled by a compound to the distance traveled by the mobile phase front in paper chromatography.

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Preparative Chromatography

A type of chromatography aimed at separating components for further use, typically for purification.

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Analytical Chromatography

A chromatography method using smaller amounts for measuring concentrations of analytes in mixtures.

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Partitioning

The separation mechanism where solutes are distributed between stationary and mobile phases.

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Eluent

The solvent responsible for carrying solutes through the chromatographic medium.

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Size Exclusion Chromatography (SEC)

A technique that separates particles based on their size as they pass through a stationary phase.

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Elution

The process by which different sized particles exit or are filtered from the stationary phase.

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Exclusion Limit

The maximum molecular weight that can still be separated in a size exclusion column.

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Permeation Limit

The minimum molecular weight that can penetrate all pores of the stationary phase.

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Eluate

The solution collected at the end of a chromatography process after filtering particles.

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Retention Time

The duration a solute remains in the column during separation.

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Molecular Weight Determination

Estimating the weight of a compound based on its elution behavior in SEC.

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Pores in SEC

Small openings in the stationary phase that allow some molecules to enter, affecting their passage.

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Separation Times

The brief duration needed for size-based separation of solutes in SEC.

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Non-Interaction Requirement

SEC needs solutes to not chemically interact with the stationary phase for accurate separation.

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Relative Retention Time (RR)

The ratio of the test substance's retention time to an internal standard.

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Paper Chromatography

A technique using paper as the stationary phase for separation.

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Thin Layer Chromatography

A method using a thin layer of adsorbent material for separation.

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Gas Chromatography

A technique that separates vaporizable compounds in a mixture.

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Ion Exchange Chromatography

A method that separates ions based on their charge.

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Choosing Solvents

The process of selecting solvents based on their polarity for chromatography.

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Affinity Chromatography

Separation based on specific interactions between a target molecule and a stationary phase.

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Electroneutrality

A state where the total charge in a solution is neutral, achieved by coupling inert charges with exchangeable counterions.

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Ionizable Molecules

Molecules that can gain or lose a charge in a solution, competing with counterions for binding.

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Gradient Elution

A method of elution where the concentration of counterions is gradually increased to separate ionized molecules.

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Step Elution

A method of elution where counterion concentrations are varied in a single step to release molecules.

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Cation Exchange Chromatography

A type of chromatography that retains positively charged molecules using negatively charged functional groups on the stationary phase.

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Moiety

A specific functional group or fragment within a molecule that contributes to its properties.

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Cationic Stationary Phase

A stationary phase that binds anions while allowing cations to pass through in chromatography.

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Anionic Stationary Phase

A stationary phase that binds cations while allowing anions to flow in chromatography.

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pH Change

Adjusting the acidity or basicity of the column to affect analyte interactions in chromatography.

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Matrix Tolerance

The capability of a chromatography method to handle complex mixtures without interference.

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Electrostatic Interactions

Forces between charged particles that attract or repel during ion exchange chromatography.

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Study Notes

Chromatography Definitions

  • Analyte: The substance being separated in chromatography.
  • Analytical Chromatography: Used to determine the presence and concentration of an analyte in a sample.
  • Bonded Phase: A stationary phase that is covalently attached to the support particles or column walls.
  • Chromatogram: The visual output of a chromatograph, showing peaks or patterns corresponding to different components.
  • Chromatograph: The equipment used for sophisticated separation, such as gas or liquid chromatography.
  • Chromatography: A physical separation method distributing components between a stationary phase and a mobile phase moving in a definite direction.
  • Eluent: The solvent that carries the analyte through the column.
  • Eluate: The mobile phase leaving the column, containing the separated components.
  • Eluotropic Series: A list of solvents ranked according to their eluting power.
  • Preparative Chromatography: Used to purify large quantities of a substance for further use, rather than analysis.
  • Retention Time: The characteristic time a particular analyte takes to travel through the system (from inlet to detector) under specific conditions.
  • Sample: The material being analyzed, which may be a single component or a mixture.
  • Solute: The sample components in partition chromatography.
  • Mobile phase: The phase that moves in a definite direction; can be liquid or gas (gas chromatography) or supercritical fluid (supercritical fluid chromatography). The mobile phase consists of the solvent.

Chromatographic Techniques

  • Types of Chromatography:
    • Chromatography is categorized by its bed, like paper chromatography, thin layer chromatography, column chromatography.
    • It is also classified based on the mechanism, like ion exchange chromatography, affinity chromatography, size exclusion chromatography, displacement chromatography, special techniques like 2-dimensional chromatography, simulated moving bed chromatography, or pyrolysis gas chromatography, fast protein liquid chromatography.
  • Gas Chromatography (GC):
    • Uses a gaseous mobile phase.
    • Usually involves a packed or capillary column with a liquid stationary phase.
    • Separates volatile compounds based on their partition equilibrium between the mobile and stationary phase.
  • High-Performance Liquid Chromatography (HPLC):
    • Uses a liquid mobile phase under high pressure.
    • Separates compounds based on interactions with a stationary phase via partition, adsorption, or ion exchange.
  • Size-Exclusion Chromatography (SEC):
    • Separates molecules primarily based on size.
    • Pores in a stationary phase of beads trap smaller molecules, allowing larger molecules to pass through faster.
  • Ion-Exchange Chromatography:
    • Separates ions and polar molecules based on their affinity for the ion exchanger.
    • The stationary phase has charged groups, attracting or repelling ions depending on their charge.
  • Paper Chromatography:
    • Involves a paper strip as a stationary phase.
    • The solvent moves up the paper, separating components based on their interaction with the paper.
  • Thin-Layer Chromatography (TLC):
    • An adsorbent material (like silica gel or alumina) coated on a plate.
    • The solvent moves up the plate, separating components based on their interaction with the stationary phase.
  • Column Chromatography: Uses a column packed with adsorbent material, to separate components based on adsorption, partition or or a combination of both.

General Chromatography Principles

  • Identification: Compounds are identified based on retention times, or reference standards, or other factors depending on the type of chromatography used.
  • Reference Substances: Used in identity tests (like TLC) to compare the ratio of movement of a substance to the solvent (Rf value) to an established standard.

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