HPLC Basics and Method Optimization
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Questions and Answers

What chromatography technique would be best to separate sodium chloride and sucrose?

  • Affinity chromatography
  • Gel filtration chromatography
  • Ion exchange chromatography (correct)
  • Reverse phase HPLC

    • Which LC mode is most appropriate to separate a mixture of caffeine, aspirin, and paracetamol?

  • Size exclusion HPLC
  • Reverse phase HPLC (correct)
  • Ion exchange HPLC
  • Normal phase HPLC

    • Which chromatographic technique exploits specific biological recognition for protein purification?

  • Partition chromatography
  • Affinity chromatography (correct)
  • Ion exchange chromatography
  • Size exclusion chromatography

    • If you want to separate amino acids, which technique would be suitable?

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

    What type of stationary phase is typically used in reverse phase HPLC?

    <p>Non-polar stationary phase</p> Signup and view all the answers

    In ion exchange chromatography, what property is primarily used for separation?

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

    Which type of chromatography would be suitable for separating chiral compounds?

    <p>Chiral chromatography</p> Signup and view all the answers

    Why is gas chromatography suitable for separating volatile organic compounds?

    <p>Because it utilizes an inert gas as the mobile phase</p> Signup and view all the answers

    What is the most appropriate chromatography technique for separating ionic compounds based on charge?

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

    In liquid chromatography, what is the purpose of using a gradient of water and a water-miscible organic solvent like acetonitrile?

    <p>To elute compounds in order of increasing hydrophobicity</p> Signup and view all the answers

    Which type of chromatography would be most suitable for separating large biomolecules based on size?

    <p>Size exclusion chromatography</p> Signup and view all the answers

    Why can cholesterol be separated by reverse phase HPLC but not by gas chromatography?

    <p>Cholesterol degrades at high temperatures</p> Signup and view all the answers

    What distinguishes normal phase liquid chromatography from reverse phase liquid chromatography?

    <p>The polarity of the stationary phase</p> Signup and view all the answers

    Which type of column would be most appropriate for separating cholesterol by reverse phase HPLC?

    <p>C18 column</p> Signup and view all the answers

    For which type of compounds is gas chromatography not suitable due to thermal instability?

    <p>Thermally labile compounds</p> Signup and view all the answers

    What role does the mobile phase play in gas chromatography?

    <p>Aiding in vaporizing the sample mixture</p> Signup and view all the answers

    What is the purpose of using isocratic elution in HPLC?

    <p>To maintain a constant mobile phase composition</p> Signup and view all the answers

    In ion exchange chromatography, what type of resin beads are commonly used as the stationary phase?

    <p>Cationic or anionic exchange resin beads</p> Signup and view all the answers

    Which type of chromatography is specifically used for separating chiral compounds?

    <p>Chiral stationary phase chromatography</p> Signup and view all the answers

    What does TLC stand for in chromatography?

    <p>Thin Layer Chromatography</p> Signup and view all the answers

    What role does the carrier gas play in gas chromatography?

    <p>Transports analytes through the column for separation</p> Signup and view all the answers

    Why is gradient elution commonly used in reverse-phase HPLC for separating polar compounds?

    <p>To improve selectivity by changing solvent polarity</p> Signup and view all the answers

    Which parameter does the van Deemter equation describe regarding chromatographic peaks?

    <p>Band broadening factors</p> Signup and view all the answers

    What type of chromatography uses porous beads made from polymers like agarose or dextran?

    <p>Size exclusion chromatography</p> Signup and view all the answers

    What does SEC stand for in chromatography when coupled with another mode like ion exchange?

    <p>Size Exclusion Chromatography</p> Signup and view all the answers

    What is the main purpose of using a chiral stationary phase in chromatography?

    <p>To selectively interact with enantiomers</p> Signup and view all the answers

    What is the primary purpose of chiral stationary phases in chromatography?

    <p>To selectively interact with one enantiomer based on different affinities</p> Signup and view all the answers

    In size exclusion chromatography (SEC), what property is primarily used for separation?

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

    What is the main difference in the mobile phase between gas chromatography (GC) and liquid chromatography (LC)?

    <p>GC uses gas mobile phase through a column; LC uses liquid mobile phase through a packed bed</p> Signup and view all the answers

    Which chromatography technique is best suited for separating inorganic ions?

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

    What is the primary factor that determines the retention of compounds in chromatography?

    <p>Phase polarity</p> Signup and view all the answers

    Which chromatography technique is preferred for separating chiral drugs?

    <p>Chiral LC using appropriate selector</p> Signup and view all the answers

    What is the primary difference between reverse phase LC and normal phase LC?

    <p>'Reverse phase uses nonpolar stationary phase; normal phase uses polar stationary phase'</p> Signup and view all the answers

    'Method validation tests parameters like linearity, range, accuracy, precision, specificity, limit of detection and quantification' primarily aim to ensure:

    <p>'Suitability of the method for intended purpose'</p> Signup and view all the answers

    'GC-MS identifies unknowns based on mass spectra and retention index' primarily enhances confidence in:

    <p>'Compound identification'</p> Signup and view all the answers

    Which chromatography technique would be most suitable for separating large biomolecules such as proteins and polymers?

    <p>Size exclusion chromatography</p> Signup and view all the answers

    What type of compounds are typically separated using reverse phase high performance liquid chromatography (HPLC)?

    <p>Polar compounds</p> Signup and view all the answers

    Which chromatography technique exploits specific biological interactions like antibody-antigen binding?

    <p>Affinity chromatography</p> Signup and view all the answers

    For which type of compounds is gas chromatography (GC) typically used as the separation method?

    <p>Volatile organic compounds</p> Signup and view all the answers

    Which type of stationary phase is most suitable for non-polar or slightly polar compounds in gas chromatography?

    <p>Dimethyl polysiloxane phase</p> Signup and view all the answers

    What is the primary factor that Size Exclusion Chromatography (SEC) uses to separate molecules?

    <p>Molecular weight</p> Signup and view all the answers

    Which chromatography technique is best suited for separating ions based on charge state?

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

    Study Notes

    Chromatography Techniques

    • Ion Exchange Chromatography: separates ions based on charge state using cation or anion exchange resins; suitable for separating amino acids, peptides, inorganic ions
      • uses cation or anion exchange resin as stationary phase
      • separates based on charge-to-size ratio
    • Reverse Phase Liquid Chromatography (HPLC): separates compounds based on hydrophobicity
      • uses non-polar stationary phase (e.g. C18) and polar mobile phase (e.g. water/acetonitrile)
      • suitable for separating moderately polar to non-polar compounds like lipids, pesticides, drugs
    • Normal Phase Liquid Chromatography (HPLC): separates compounds based on polarity
      • uses polar stationary phase (e.g. silica) and non-polar mobile phase (e.g. hexane/ethyl acetate)
      • suitable for separating polar compounds like alcohols, amines, acids
    • Size Exclusion Chromatography (SEC): separates compounds based on size/molecular weight
      • uses porous beads that selectively retard passage of larger molecules
      • suitable for separating polymers, proteins, oligosaccharides by molecular weight
    • Affinity Chromatography: separates compounds based on biological interactions
      • exploits specific biological recognition between protein and ligand attached to stationary phase
      • suitable for purifying target biomolecules from complex mixtures

    Applications of Chromatography

    • Volatile Organic Compounds (VOCs): Gas Chromatography (GC) is typically used
    • Thermally Labile Compounds: Liquid Chromatography (LC) techniques like HPLC are preferred
    • Polar Compounds: Reverse Phase HPLC or Normal Phase HPLC can be used
    • Large Biomolecules: Size Exclusion Chromatography (SEC) is suitable
    • Chiral Separations: Chiral stationary phases in GC or HPLC can be used
    • Ionizable Compounds: Ion Exchange Chromatography separates based on charge state
    • Inorganic Ions: Ion Chromatography uses different eluents and stationary phases tailored for separating anions/cations
    • Metal Complexes: Specialized techniques like TLC may be required

    Gas Chromatography (GC)

    • Column: long, narrow capillary tubing coated internally with a thin film of stationary phase
    • Mobile Phase: inert gas like helium
    • Separation: based on differences in volatility and affinity between analytes and stationary phase
    • Detection: usually by Flame Ionization Detector (FID) or Mass Spectrometer (MS)

    High Performance Liquid Chromatography (HPLC)

    • Column: contains tiny porous particles packed into a stainless steel tube
    • Mobile Phase: starts with water then increases proportion of water-miscible organic solvent like acetonitrile or methanol
    • Separation: based on interactions between analytes and mobile/stationary phases
    • Detection: usually by UV/Vis or Mass Spectrometer (MS)

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    Description

    Learn about the fundamentals of High Performance Liquid Chromatography (HPLC), including the use of mobile phases, sample injection, elution modes, and detection methods. Discover how method optimization involves finding the optimal mobile phase composition and flow rate to achieve accurate results.

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