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Chromatography Basics
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Chromatography Basics

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Questions and Answers

What is the purpose of the eluent in chromatography?

  • To carry the analyte through the column (correct)
  • To detect the analyte
  • To separate the components of the mixture
  • To purify the sample

    • What is the purpose of chromatography in pharmaceutical industry?

  • To identify the components of a mixture
  • To analyze the components of a mixture
  • To determine the amount of each chemical found in a new product (correct)
  • To separate the components of a mixture

    • What is the term for the fluid that exits the column?

  • Eluent
  • Solvent
  • Mobile phase
  • Eluate (correct)

    • What is linear velocity in chromatography?

    <p>The distance traveled by the mobile phase per minute</p> Signup and view all the answers

    What is the retention time in chromatography?

    <p>The time it takes for the analyte to pass through the system</p> Signup and view all the answers

    What is the primary use of chromatography in scientific research?

    <p>To analyze and identify the components of a mixture</p> Signup and view all the answers

    Which of the following organizations may use chromatography to detect blood or alcohol levels in a patient's bloodstream?

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

    What is the primary purpose of Gas Chromatography?

    <p>To separate vaporized samples with a carrier gas</p> Signup and view all the answers

    What is the function of the antibody in Radioimmunoassay (RIA)?

    <p>To bind to the antigen of interest</p> Signup and view all the answers

    What is the purpose of the standard curve in Radioimmunoassay (RIA)?

    <p>To plot the radioactivity against known concentrations of the antigen</p> Signup and view all the answers

    Which of the following types of chromatography separates dried liquid samples with a liquid solvent and a paper strip?

    <p>Paper Chromatography</p> Signup and view all the answers

    What is the primary purpose of Radioimmunoassay (RIA)?

    <p>To measure the concentration of a specific antigen or hormone in a sample</p> Signup and view all the answers

    What is the function of the radiolabeled antigen (tracer) in Radioimmunoassay (RIA)?

    <p>To compete with the antigen of interest for antibody binding</p> Signup and view all the answers

    Which of the following types of chromatography separates liquid samples with a liquid solvent and a column composed of solid beads?

    <p>Liquid Chromatography</p> Signup and view all the answers

    Which of the following fields is NOT commonly associated with RIA?

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

    What is a limitation of RIA?

    <p>Radioactive material handling requirements</p> Signup and view all the answers

    What is the primary purpose of PCR?

    <p>To amplify specific DNA sequences</p> Signup and view all the answers

    What is the role of primers in PCR?

    <p>To bind to the target DNA sequence</p> Signup and view all the answers

    What is the temperature range for the annealing step in PCR?

    <p>50-65°C</p> Signup and view all the answers

    What is the enzyme responsible for synthesizing new DNA strands in PCR?

    <p>Taq polymerase</p> Signup and view all the answers

    What is the result of the denaturation step in PCR?

    <p>The melting of the DNA double helix into single strands</p> Signup and view all the answers

    Who developed the PCR technique?

    <p>Kary Mullis</p> Signup and view all the answers

    What is the ideal temperature for human polymerase to work?

    <p>37°C</p> Signup and view all the answers

    In which direction does the Taq polymerase add DNA bases to the single strand?

    <p>5’ to 3’</p> Signup and view all the answers

    Which type of PCR amplifies RNA sequences?

    <p>Reverse Transcription PCR (RT-PCR)</p> Signup and view all the answers

    What is a limitation of PCR?

    <p>Contamination risk</p> Signup and view all the answers

    What is the purpose of heating the reaction mixture to 94-95°C in PCR?

    <p>To break the hydrogen bonds between the bases in the two strands of template DNA</p> Signup and view all the answers

    What is the function of the primers in PCR?

    <p>To provide a short region of double-stranded DNA for the polymerase enzyme to work with</p> Signup and view all the answers

    What is the purpose of an enzyme in an enzyme immunoassay?

    <p>To allow for detection through color changes</p> Signup and view all the answers

    What is the primary application of EIAs in medical research?

    <p>All of the above</p> Signup and view all the answers

    What is the temperature range for the annealing step in PCR?

    <p>50-65°C</p> Signup and view all the answers

    How many primers are used in PCR?

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

    What is the advantage of PCR in terms of time?

    <p>Takes several hours to complete</p> Signup and view all the answers

    Why is the Taq DNA polymerase enzyme used in PCR?

    <p>Because it can tolerate temperatures above 80°C</p> Signup and view all the answers

    What is the result of the denaturation step in PCR?

    <p>Two single strands of DNA that will act as templates to produce new copies of each strand</p> Signup and view all the answers

    Which type of PCR monitors amplification in real-time?

    <p>Real-time PCR (qPCR)</p> Signup and view all the answers

    What is the purpose of the extension step in PCR?

    <p>To add DNA bases to the new strand</p> Signup and view all the answers

    What is the temperature used in the extension step of PCR?

    <p>72°C</p> Signup and view all the answers

    Study Notes

    Chromatography

    • Chromatogram is the visual output of a chromatograph, which separates mixtures into individual components.
    • Chromatograph is equipment that enables sophisticated separation of mixtures.
    • Retention time is the characteristic time it takes for a particular analyte to pass through the system under set conditions.
    • Eluent is the solvent that carries the analyte, while eluate is the fluid exiting the column.
    • Elution is the process of passing the mobile phase through the column.
    • Flow rate is the measure of how much mobile phase passes through the column per minute (ml/min).
    • Linear velocity is the distance passed by the mobile phase per minute in the column (cm/min).

    Uses of Chromatography

    • Analyze: Examine a mixture, its components, and their relations to one another.
    • Identify: Determine the identity of a mixture or components based on known components.
    • Purify: Separate components to isolate one of interest for further study.
    • Quantify: Determine the amount of the mixture and/or components present in the sample.
    • Real-life examples: Pharmaceutical, hospital, law enforcement, and environmental agency applications.

    Types of Chromatography

    • Liquid Chromatography: Separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase).
    • Gas Chromatography: Separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or solid beads (stationary phase).
    • Paper Chromatography: Separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase).
    • Thin-Layer Chromatography: Separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase).

    Radioimmunoassay (RIA)

    • RIA is a laboratory technique used to measure the concentration of a specific antigen or hormone in a sample.
    • It is a highly sensitive and specific method that uses radioactivity to detect and quantify the binding of antibodies to antigens.
    • Principle: Preparation, incubation, separation, measurement, and calibration.
    • Widely used in various fields, including endocrinology, immunology, infectious disease diagnosis, and cancer research.
    • Limitations: Radioactive material handling requirements, limited shelf life of radiolabeled tracers, and potential for non-specific binding.

    Polymerase Chain Reaction (PCR)

    • PCR is a laboratory technique used to make many copies of a specific DNA sequence.
    • Steps: Denaturation, annealing, extension, and repeat.
    • PCR is a powerful tool in molecular biology, used for DNA cloning, genetic testing, and forensic analysis.
    • PCR has five core 'ingredients': DNA template, primers, DNA nucleotide bases, Taq polymerase enzyme, and a buffer.
    • Types of PCR: Conventional PCR, Real-time PCR (qPCR), Reverse Transcription PCR (RT-PCR), and Multiplex PCR.
    • Applications: Genetic testing, gene expression analysis, forensic analysis, cancer research, and viral load detection.
    • Advantages: High sensitivity, high specificity, and fast results.
    • Limitations: Contamination risk and inhibitors.

    Enzyme Immunoassay (EIA)

    • EIA is a type of immunoassay that uses an enzyme bonded to an antigen or antibody to detect specific molecules in a sample.
    • Types of EIA: Enzyme-linked immunosorbent assay (ELISA), Enzyme multiplied immunoassay technique (EMIT), Fluorescent enzyme immunoassays (FEIAs), Chemiluminescent immunoassays (CLIAs), and Radioimmunoassays (RIAs).
    • EIAs are widely used in medical and research applications for measuring protein, pathogens, and other molecules in blood samples.

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    Description

    Learn about the fundamental concepts of chromatography, including chromatograms, chromatographs, retention time, eluent, eluate, and elution.

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