Chromatography Separation Methods
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Questions and Answers

What is the primary difference between open column chromatography and other types of column chromatography?

  • The shape of the column
  • The type of stationary phase used
  • The use of atmospheric pressure (correct)
  • The material used to pack the column

    • What is the purpose of adding glass wool or filter paper to the top of the column?

  • To increase the surface area
  • To decrease the pressure
  • To filter out impurities
  • To protect the column from disturbance (correct)

    • What is the main criterion for selecting a method of packing the column?

  • The density of the solid (correct)
  • The shape of the column
  • The type of stationary phase
  • The flow rate of the mobile phase

    • What is the characteristic of isocratic elution?

    <p>Addition of solvent mixture of fixed composition</p> Signup and view all the answers

    What is the difference between step-wise and gradient elution?

    <p>Step-wise elution involves a sudden change, while gradient elution involves a continuous change</p> Signup and view all the answers

    What is the role of the support material in partition column chromatography?

    <p>To provide a surface for the stationary phase</p> Signup and view all the answers

    What is the primary difference between adsorption and partition chromatography?

    <p>Theoretical significance only</p> Signup and view all the answers

    Why is it important to avoid air bubbles during packing?

    <p>To prevent channeling</p> Signup and view all the answers

    What is the purpose of the stationary phase in column chromatography?

    <p>To separate the components of the mixture</p> Signup and view all the answers

    What is the characteristic of the mobile phase in gradient elution?

    <p>It has a continuous change in composition throughout the process</p> Signup and view all the answers

    What is the main function of the stationary phase in column chromatography?

    <p>To separate the components of the mixture</p> Signup and view all the answers

    What is the purpose of packing the column in column chromatography?

    <p>To provide a surface for the mobile phase to interact with</p> Signup and view all the answers

    What is the characteristic of the mobile phase in isocratic elution?

    <p>It remains constant in composition</p> Signup and view all the answers

    What is the purpose of applying the sample evenly to the top of the column?

    <p>To prevent disturbance of the column</p> Signup and view all the answers

    What is the main difference between partition and adsorption chromatography?

    <p>Only theoretical significance</p> Signup and view all the answers

    What is the purpose of using a glass column in traditional column chromatography?

    <p>To hold the stationary phase in place</p> Signup and view all the answers

    What is the effect of air bubbles in the column during packing?

    <p>It decreases the surface area of the stationary phase</p> Signup and view all the answers

    What is the characteristic of gradient elution in column chromatography?

    <p>The mobile phase changes composition continuously</p> Signup and view all the answers

    What is the purpose of using a wet, dry, or slurry method for packing the column?

    <p>To select the best method based on the density of the solid</p> Signup and view all the answers

    What is the purpose of the support material in partition column chromatography?

    <p>To hold the stationary phase in place</p> Signup and view all the answers

    What is the primary difference between random error and systematic error in analytical data?

    <p>Random error is unpredictable and affects the precision of a method, whereas systematic error is predictable and affects the accuracy of a method.</p> Signup and view all the answers

    What is the purpose of assessing the quality of analytical methods?

    <p>To evaluate the precision, accuracy, sensitivity, and specificity of a method to ensure reliable results.</p> Signup and view all the answers

    What is the definition of accuracy in the context of analytical methods?

    <p>The closeness of the mean of a set of replicate analyses to the true value of the sample.</p> Signup and view all the answers

    What is the difference between precision and accuracy?

    <p>Precision refers to the closeness of replicate measurements to each other, whereas accuracy refers to the closeness of a measured value to a standard or known value.</p> Signup and view all the answers

    What is the definition of sensitivity in the context of analytical methods?

    <p>The ability of a method to detect small amounts of the test substance.</p> Signup and view all the answers

    Why is it important to consider both precision and accuracy in evaluating the quality of analytical methods?

    <p>Because precision and accuracy are independent of each other, and a method can be precise but not accurate, or accurate but not precise.</p> Signup and view all the answers

    What is the purpose of quality control in analytical biochemistry?

    <p>To ensure that the analytical methods and results are reliable and accurate.</p> Signup and view all the answers

    What is the difference between quality control and quality assessment?

    <p>Quality control refers to the procedures and processes used to ensure quality, whereas quality assessment refers to the evaluation of the quality of analytical results.</p> Signup and view all the answers

    Why is it important to have standard operating procedures in analytical laboratories?

    <p>To ensure that the analytical methods and procedures are consistent and reproducible, and to minimize errors and variability.</p> Signup and view all the answers

    What is the purpose of accreditation of laboratories?

    <p>To ensure that the laboratory meets the required standards and criteria for analytical quality and reliability.</p> Signup and view all the answers

    Study Notes

    Separation Methods

    • Separation is crucial in analysis of biological samples to avoid interference from other substances
    • Two main approaches to separation: isolate the test substance or remove the interfering substance

    Principles of Separation Techniques

    • Separation techniques are based on differences in molecular characteristics such as polarity, ionic nature, size, and shape
    • These differences affect the interaction between molecules and the stationary and mobile phases

    Classification of Separation Techniques

    • Based on molecular characteristics:
      • Polarity: Gas-liquid chromatography, Liquid-liquid chromatography, Liquid-solid chromatography
      • Ionic nature: Ion-exchange chromatography, Electrophoresis
      • Size (mass): Gel permeation chromatography, Dialysis, Ultracentrifugation
      • Shape: Affinity chromatography

    Chromatography

    • A technique used to separate and identify the components of a mixture
    • Works by allowing molecules to distribute between a stationary and mobile medium
    • Molecules that spend more time in the mobile phase are carried along faster

    Thin Layer Chromatography (TLC)

    • Uses a stationary phase (adsorbent) and a mobile phase (solvent)
    • The sample is spotted on the plate and the solvent is allowed to move up the plate
    • Components of the mixture are separated based on their interactions with the stationary and mobile phases
    • Visualization techniques are used to identify the separated components, such as UV light, alkaloids, cardiac glycosides, sugars, and amino acids

    Interpreting the Data

    • Rf (retention factor) values are calculated for each spot and are characteristic for a given compound
    • Rf values can be used to identify unknown substances by comparing them to known values
    • Purity of a sample can be estimated from the chromatogram

    Paper Chromatography

    • A method of partition chromatography using filter paper as the carrier
    • The factor governing separation is the partition between two immiscible phases
    • General procedure involves choice of paper and solvent, desalting of sample, application of the sample, equilibration of paper, development, detection, and identification of substances

    Multiple Chromatography

    • Includes procedures where development is repeated after one development is completed
    • A- multiple development: repeated development in the same direction with the same or different solvent systems
    • B- two-dimensional chromatography: development in a direction perpendicular to the first, with a different solvent system

    Column Chromatography (CC)

    • A chromatographic method where the stationary phase is packed into a column and the mobile phase is a moving liquid or gas
    • Stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or gravity

    Open Column Chromatography

    • Traditional column chromatography characterized by addition of mobile phase under atmospheric pressure
    • Packing and operating the column involve packing, sample application, and elution techniques

    Separation Methods

    • The importance of separation in biological samples lies in the presence of substances that may interfere with the analysis or affect the quality of the results.
    • Separation is necessary to either isolate the test substance or remove the interfering substances before analysis.

    Principles of Separation Techniques

    • Separation techniques are based on molecular characteristics, such as polarity, ionic nature, size, and shape.
    • These characteristics are used to separate biomolecules based on their physical properties.

    Classification of Separation Techniques

    • Separation techniques can be classified based on molecular characteristics, including:
      • Polarity: volatility, solubility, adsorptivity
      • Ionic nature: charge
      • Size (mass): diffusion, sedimentation
      • Shape: ligand binding

    Chromatography

    • Chromatography is a technique used to separate and identify the components of a mixture.
    • It works by distributing molecules between a stationary and a mobile phase.
    • Molecules that spend most of their time in the mobile phase are carried along faster.
    • Chromatography is a physical method of separation that uses a stationary phase and a mobile phase to separate components based on their distribution constants.

    Thin Layer Chromatography (TLC)

    • TLC is a method for identifying substances and testing the purity of compounds.
    • It is a useful technique because it is relatively quick and requires small quantities of material.
    • The stationary phase is a thin layer of adsorbent coated on a plate.
    • The mobile phase is a developing liquid that travels up the stationary phase, carrying the samples with it.
    • Components of the sample will separate on the stationary phase according to their adsorption and solubility properties.
    • TLC is used to identify compounds by spotting known substances next to unknown substances on the same plate.
    • The purity of a sample can be estimated from the chromatogram, with impure samples often developing as two or more spots, and pure samples showing only one spot.

    Paper Chromatography

    • Paper chromatography is a method of partition chromatography that uses filter paper strips as a carrier or inert support.
    • The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases.
    • One phase is usually water adsorbed on cellulose fibers in the paper (stationary phase), and the second is the organic solvent (mobile phase) that flows past the sample on the paper.

    Multiple Chromatography

    • Multiple chromatography includes all procedures in which the development is repeated after one development is completed.
    • Examples include:
      • A- multiple development: repeated development in the same direction to resolve substances with close Rf values.
      • B- two-dimensional chromatography: development in a direction perpendicular to the first, with a solvent system different from that used initially.

    Column Chromatography (CC)

    • Column chromatography includes chromatographic methods in which the stationary phase is packed into a column.
    • The mobile phase is a moving liquid or gas that is forced under pressure or gravity.
    • Stationary phase is held in a narrow tube through which the mobile phase is forced.

    Open Column Chromatography

    • Traditional column chromatography is characterized by addition of mobile phase under atmospheric pressure.
    • The stationary phase is packed in a glass column.

    Packing and Operating the Column

    • Packaging methods depend on the density of the solid, and techniques used include wet, dry, and slurry methods.
    • Avoid inclusion of air bubbles.
    • Sample application involves adding the sample evenly and in a concentrated solution to the top of the column, which is protected from disturbance.
    • Elution techniques include:
      • Isocratic elution: addition of solvent mixture of fixed composition during the whole process.
      • Gradient elution: continuous or linear elution, in which there is a continuous change in the composition of the mobile phase over a period of time.
      • Step-wise or fractional elution: in which the change is not continuous, with a sudden change in the composition of the mobile phase followed by a period where the mobile phase is held constant.

    Errors in Analytical Data

    • Errors can be divided into two types: Random Error and Systematic Error
    • Random Error:
      • Caused by unpredictable changes during an experiment
      • Affects measurements differently each time
      • Comes from unpredictable changes during an experiment
      • Examples: reading a volume from a different angle each time, variable volume delivered by auto pipettes
    • Systematic Error:
      • Caused by instrumental factors or error of the methods
      • Affects measurements the same amount or by the same proportion
      • Examples: instrumental factors (measuring length with a metal ruler), error of the methods (not setting an instrument to zero)
    • Both Random Error and Systematic Error affect the accuracy of results, but Systematic Error can be predicted and avoided

    Quality of Data

    • Variability refers to the spread out of a set of data
    • Variability is due to two types of errors: Random Error and Systematic Error
    • Importance of quality of data: to minimize errors

    Assessment of Analytical Methods

    • Analytical methods should be:
      • Precise
      • Accurate
      • Sensitive
      • Specific
    • Assessment of analytical methods:
      • Accuracy: the closeness of the mean of a set of replicate analyses to the true value of the sample
      • Precision: the extent to which a number of replicate measurements of a sample agree with one another
      • Sensitivity: the ability to detect small amounts of the test substance

    Quality Assurance in Analytical Biochemistry

    • Quality control: implementing procedures to ensure quality of data
    • Quality assessment: evaluating the quality of data
    • Accreditation of laboratories: ensuring that laboratories meet certain standards

    Objectives

    • Importance of quality of data
    • Type of errors
    • Assessment of analytical methods

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    Description

    This quiz covers the importance of separation in chromatography, including Thin Layer Chromatography (TLC), Paper Chromatography (PC), and Column Chromatography (CC). It also discusses the difficulties in analyzing biological samples.

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