Chromatography Techniques Overview
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Questions and Answers

What is the primary purpose of the final column washing step in liquid chromatography?

  • To change the buffer composition.
  • To strip remaining compounds.
  • To prepare the column to be reused. (correct)
  • To elute strongly interacting molecules.

    • In isocratic liquid chromatography, the mobile phase solvent composition changes over time.

    False (B)

    What buffer is used to saturate the column for subsequent reuse after stripping the remaining compounds?

    equilibration buffer

    Gradient elution is best suited for analyzing ______ separations.

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

    Match the elution type to its description:

    <p>Isocratic Elution = Mobile phase solvent composition remains constant with time Gradient Elution = Mobile-phase solvent composition increases with time</p> Signup and view all the answers

    What does the separation factor represent in chromatography?

    <p>The ratio of the K' for two peaks (D)</p> Signup and view all the answers

    The efficiency of a chromatographic system is independent of the width of the peaks.

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

    What is the relationship between HETP, column length (L), and the number of theoretical plates (N)?

    <p>H = L/N</p> Signup and view all the answers

    In gas chromatography, molecules are separated based on their boiling points and their interaction with the __________ phase.

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

    Match the following chromatographic parameters with their descriptions:

    <p>Separation factor = Ratio of K’ for 2 peaks Efficiency (N) = Number of theoretical plates HETP (H) = Height equivalent of theoretical plates Resolution (RS) = Separation between two peaks</p> Signup and view all the answers

    Which of the following is NOT a factor that contributes to band spreading in chromatography?

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

    Gas chromatography can only have a gas-liquid stationary phase.

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

    In gas chromatography, what is the role of the inert gas?

    <p>To sweep the sample across the solid phase. (B)</p> Signup and view all the answers

    The stationary phase in gas chromatography is a gas.

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

    What is the primary use for gas chromatography?

    <p>analytical work</p> Signup and view all the answers

    The sample in gas chromatography is introduced onto the column as a "plug" of ___________.

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

    Match the column types with their descriptions:

    <p>Packed column = Contains solid support material coated with liquid stationary phase Wall-coated open tubular (WCOT) column = Capillary tube whose walls are coated with liquid stationary phase Support-coated open tubular (SCOT) column = Capillary tube lined with support material onto which the stationary phase is adsorbed</p> Signup and view all the answers

    What is the temperature of the sample injector usually?

    <p>50°C higher than the boiling point of the least volatile component. (D)</p> Signup and view all the answers

    SCOT columns are generally more efficient than WCOT columns.

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

    Name one common carrier gas used in gas chromatography.

    <p>nitrogen, helium, argon, or carbon dioxide</p> Signup and view all the answers

    The mobile phase in gas chromatography is a ___________.

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

    What is the maximum pressure at which HPLC is typically conducted?

    <p>5,000 psi (B)</p> Signup and view all the answers

    HPLC is suitable for affinity chromatography.

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

    What does ELSD stand for?

    <p>Evaporative light scattering detector</p> Signup and view all the answers

    HPLC can be used to separate molecules based on size, hydrophobicity, or ________.

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

    Match the chromatography step with its description:

    <p>Resin Equilibration = A buffer compatible with the molecule of interest is passed over the column Sample Loading = Sample is loaded onto the column Column Washing = Molecules that interact weakly with the resin are removed</p> Signup and view all the answers

    Which of the following detectors is commonly used in HPLC?

    <p>UV-absorbance detector (B)</p> Signup and view all the answers

    HPLC can detect concentrations down to parts per million.

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

    What does SEC stand for in the context of chromatography?

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

    The combination of HPLC and mass spectrometry is abbreviated as ________.

    <p>LC-MS</p> Signup and view all the answers

    Which of the following best describes a typical first step in a liquid chromatography protocol?

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

    What provides strength to the thin walls of capillary columns?

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

    Mass flow dependent detectors do not destroy the sample.

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

    What is the typical elution time when the column temperature is slightly above the average boiling point of the sample?

    <p>2 - 30 minutes</p> Signup and view all the answers

    A __________ detector responds to all compounds except the carrier gas.

    <p>non-selective</p> Signup and view all the answers

    Match the detector type with the appropriate description:

    <p>Non-selective detector = Responds to all compounds except the carrier gas Selective detector = Responds to a range of compounds with a common physical or chemical property Specific detector = Responds to a single chemical compound Mass flow dependant detector = Signal is related to the rate at which solute molecules enter the detector</p> Signup and view all the answers

    What is a key advantage of using a Flame Ionization Detector (FID)?

    <p>It is mass sensitive rather than concentration sensitive. (A)</p> Signup and view all the answers

    Minimal temperatures in gas chromatography help with good resolution, but they decrease elution times.

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

    What support gases are used in Flame Ionization Detectors?

    <p>Hydrogen and air</p> Signup and view all the answers

    If a sample has a wide boiling range, then _________ programming can be useful.

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

    Which of these is a characteristic of the flexible capillary columns?

    <p>They have low reactivity. (B)</p> Signup and view all the answers

    Study Notes

    Chromatography Overview

    • Chromatography is a technique for separating mixtures into their components, allowing for analysis, identification, purification, and quantification.
    • It has various applications, including analysis, identification, purification, and quantification.

    Column Chromatography

    • Separates molecules in complex mixtures.
    • Stationary phase (resin) is packed into a column.
    • Mobile phase is passed through the column to achieve separation.
    • Separation purpose can be analytical or preparative.
    • Gas and liquid chromatography are performed on columns.

    Column Chromatography Systems

    • Performed on a packed 3-dimensional stationary phase inside a glass, plastic, or metal column.
    • Used for both preparative and analytical purposes.

    Gas Chromatography (GC)

    • Mobile phase is a gas.
    • Gas-Liquid (liquid-coated support) or Gas-Solid (solid support) stationary phases.
    • Separates molecules based on boiling points and interactions with stationary phase.
    • Heated to vaporize the least volatile component and swept across the stationary phase by inert gas, thus gas chromatography.
    • Stationary phase is a liquid or polymer coated inside a long capillary column through which the gas mixture flows.
    • Typically used for analytical work, although preparative uses exist.

    GC Instrumental Components

    • Carrier gas (e.g., nitrogen, helium, argon, carbon dioxide) - inert.
    • Sample injection port to introduce sample in vapor form; high enough temperature to vaporize the sample.
    • Column oven to control temperature.
    • Detector to measure separated components (e.g., flame ionization detector (FID), thermal conductivity detector (TCD)).

    GC Detectors

    • Different detectors provide specific selectivity.
    • Non-selective detectors respond to all compounds (except the carrier gas).
    • Selective detectors respond to compounds with similar physical/chemical properties.
    • Specific detectors respond to a single compound.
    • Signal from concentration-dependent detectors is related to the concentration of solute in the detector.
    • Signal from mass flow-dependent detectors is related to the rate of solute entering the detector.
    • Flame ionization detector (FID) is mass-sensitive, giving robust readings unaffected by flow rate changes.

    Liquid Chromatography (LC)

    • Separates dissolved molecules in a liquid mobile phase over a solid stationary phase.
    • Individual components move through the stationary phase at different rates due to varying interactions.

    LC Types

    • Planar liquid chromatography (e.g., paper chromatography, thin-layer chromatography (TLC)).
    • Column liquid chromatography.

    LC Stationary Phases

    • Ion exchange
    • Size exclusion (gel filtration)
    • Hydrophobic interaction chromatography (HIC)
    • Partition chromatography
    • Adsorption chromatography
    • Multimodal/mixed-mode chromatography (MMC)

    LC Instrumental Components

    • Mobile phase is a liquid, and the stationary phase is one or more media components.
    • Type of column and mobile phase depends on properties of molecules of interest.

    LC Workflow

    • Resin equilibration (compatible buffer for the resin).
    • Sample loading (manually or using a pump).
    • Column washing (remove non-specifically interacting molecules).
    • Sample elution (separate molecules with different interaction strengths) using buffer composition changes.
    • Final column washing (reuse column for future separations).

    Gradient vs Isocratic conditions

    • Isocratic: Constant mobile phase composition; Simple, quality control procedures
    • Gradient: Mobile phase composition changes over time; separation of complex mixtures.

    UPLC/UHPLC (Ultra Performance/Ultra High Performance Liquid Chromatography):

    • Operate at high pressures (up to 15,000-20,000 psi) to achieve rapid separations, compared to standard HPLC.
    • Improves resolution
    • Useful for complex mixtures

    XBridge Columns

    • Suitable for high pH, high temperatures, volatile eluents.

    HPLC Considerations

    • Resolution: Separation of peaks in a chromatogram
    • Yield: Amount of desired compound recovered
    • Sample integrity: Important for preparative chromatography
    • Sample purity: Important property for downstream applications
    • Absolute purity: Critical for specific applications such as antibody production.

    Chromatography and pH

    • Selectivity of different types of chromatography resins changes at different pH. For example, some resins are better at high pH while others are better at low pH.

    Chromatography Polarity

    • Polarity of molecules influences separation in chromatography.
    • Polar molecules interact better with polar stationary phases, and vice versa for non-polar molecules

    Normal and Reversed phase Chromatography

    • Normal phase: Polar stationary phase/non-polar mobile phase.
    • Reversed phase: non-polar stationary phase/polar mobile phase- most common method.

    Chromatography Media Selection

    • Select columns that do not require buffer exchanges or concentration steps whenever possible.
    • Sample purity, integrity, and yield interact, frequently in an inverse relationship.
    • Consider salt precipitation from solution (salt-out) when selecting HIC.

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    Chromatography PDF

    Description

    Explore the fundamental concepts and applications of chromatography, including both column and gas chromatography. This quiz covers separation methods, stationary and mobile phases, and various analytical and preparative techniques. Test your understanding of these essential laboratory techniques.

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