Practical Spectral Analysis - CP508
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Questions and Answers

Which of the following factors does NOT affect the rate of migration of an ion in an electrical field?

  • Strength of electrical field
  • Net charge of molecule
  • Color of the particle (correct)
  • Solvent temperature

How is mobility of particles affected by the shape and size of the particle?

  • Size and shape have no effect on migration
  • Larger particles migrate faster than smaller ones
  • Shape does not influence migration velocity
  • Smaller particles migrate faster due to less friction (correct)

What happens to the mobility of a molecule at its zwitterion state?

  • Mobility increases significantly
  • Mobility is doubled
  • Mobility becomes negative
  • Mobility is retarded to zero (correct)

Which type of electrophoresis involves the migration of charged particles on supporting media?

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

Which of the following types of electrophoresis relies on the pH of the solvent to influence migration?

<p>Isoelectric Focusing (A)</p> Signup and view all the answers

What does the strength of the electrical field in electrophoresis determine?

<p>The velocity of particle movement (C)</p> Signup and view all the answers

Which of the following describes the behavior of ions in electrophoresis?

<p>They move towards opposite charges. (A)</p> Signup and view all the answers

In the context of mobility in electrophoresis, what role do buffers play?

<p>To set the pH, influencing the charge of molecules (C)</p> Signup and view all the answers

What is one application of molecular cloning mentioned in the content?

<p>Creating new genetic circuits (A)</p> Signup and view all the answers

Which technique can be utilized for DNA fingerprinting?

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

What type of gel is more robust than agarose gels?

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

How does PAGE separate proteins during the electrophoresis process?

<p>By charge to mass ratio and molecular size (D)</p> Signup and view all the answers

What effect does the electric field have in the PAGE procedure?

<p>Drives macromolecular anions towards the anode (A)</p> Signup and view all the answers

What is a benefit of polyacrylamide gels mentioned in the content?

<p>They maintain stability over a wide range of pH and temperature (B)</p> Signup and view all the answers

What is the purpose of placing the sample over the gel column during the PAGE procedure?

<p>To allow for electrophoretic mobility (B)</p> Signup and view all the answers

Which characteristic differentiates polyacrylamide gels from agarose gels?

<p>Covalent cross-linking structure (A)</p> Signup and view all the answers

What is the typical pressure range that the pump can achieve?

<p>4000 psi (A)</p> Signup and view all the answers

What is the purpose of the sample injector in the HPLC system?

<p>To introduce the liquid sample into the flow stream (C)</p> Signup and view all the answers

Which factor does NOT affect the sensitivity of an HPLC column?

<p>Flow rate of the mobile phase (D)</p> Signup and view all the answers

What is a common particle size range for stationary phases used in HPLC?

<p>3–10 µm (D)</p> Signup and view all the answers

What is the significance of maintaining a constant temperature during HPLC analysis?

<p>To enhance the stability of the mobile phase and column (A)</p> Signup and view all the answers

What is the typical internal diameter range for standard HPLC columns?

<p>2 - 5 mm (C)</p> Signup and view all the answers

Why are smaller particle sizes often preferred in traditional HPLC?

<p>They provide more surface area and better separations (C)</p> Signup and view all the answers

How does the pore size of the stationary phase influence analyte interaction?

<p>Pore size determines the ability of analyte molecules to penetrate and interact with the stationary phase (A)</p> Signup and view all the answers

What is the main advantage of using Direct ELISA over other methods?

<p>Fewer steps and elimination of cross-reactivity from secondary antibodies (A)</p> Signup and view all the answers

In an Indirect ELISA, what is added to the plate after washing away unbound primary antibodies?

<p>A secondary antibody conjugated to an enzyme (C)</p> Signup and view all the answers

What role does the substrate play in an Indirect ELISA?

<p>It is converted by the enzyme to produce a colored product (B)</p> Signup and view all the answers

Which of the following statements reflects a feature of Competitive ELISA?

<p>The antigen-antibody mixture is filled into an antigen-coated well (B)</p> Signup and view all the answers

Why is Blocking important in the Indirect ELISA procedure?

<p>To prevent false positive results (A)</p> Signup and view all the answers

What is a key advantage of Indirect ELISA compared to Direct ELISA?

<p>It has increased sensitivity by using multiple labeled antibodies (A)</p> Signup and view all the answers

What is primarily measured using a spectrophotometric plate reader in Indirect ELISA?

<p>The colored product produced from enzyme-substrate reaction (B)</p> Signup and view all the answers

Which feature is NOT an advantage of using Indirect ELISA?

<p>Ability to conduct the assay at low temperatures (D)</p> Signup and view all the answers

What is the primary function of the fluidics system in a flow cytometer?

<p>To transport the sample to the flow cell (A)</p> Signup and view all the answers

Which component of a flow cytometer is responsible for measuring Forward Scatter (FSC)?

<p>A detector positioned in line with the light beam (D)</p> Signup and view all the answers

What role does the electronics system play in a flow cytometer?

<p>It digitizes and processes the photocurrent (C)</p> Signup and view all the answers

How does Side Scatter (SSC) contribute to the analysis of particles in flow cytometry?

<p>It reflects the complexity of the cell's nuclear structure (D)</p> Signup and view all the answers

What is the purpose of using fluorescent labels in flow cytometry?

<p>To detect different cell types based on emitted light (A)</p> Signup and view all the answers

What aspect of a cell can the Forward Scatter (FSC) measurement indicate?

<p>The cell's volume (D)</p> Signup and view all the answers

What is the result of scattering light by suspended particles in a flow cytometer?

<p>It helps in identifying and analyzing cell properties (C)</p> Signup and view all the answers

Which of the following components is NOT part of the optical system in flow cytometry?

<p>Fluid transport tubes (C)</p> Signup and view all the answers

What is the main purpose of isoelectric focusing in protein analysis?

<p>To determine the isoelectric point (PI) of proteins (B)</p> Signup and view all the answers

At what pH does a protein stop migrating during isoelectric focusing?

<p>When the protein reaches the isoelectric pH (B)</p> Signup and view all the answers

In HPLC, what role does the stationary phase play?

<p>It separates components based on their affinity (D)</p> Signup and view all the answers

What is the primary characteristic of compounds in HPLC during the separation process?

<p>They are attracted by both the mobile and stationary phases (A)</p> Signup and view all the answers

What happens to a protein at a pH below its isoelectric point?

<p>It migrates towards the cathode due to positive charge (B)</p> Signup and view all the answers

What is a chromatogram in the context of chromatography?

<p>The visual output representing the separated components (A)</p> Signup and view all the answers

Which of the following statements about proteins in isoelectric focusing is true?

<p>Proteins stop moving after reaching their isoelectric charge (C)</p> Signup and view all the answers

Which parameter is essential for HPLC to effectively separate components?

<p>The differential affinities for the phases (B)</p> Signup and view all the answers

Flashcards

Molecular Cloning

A technique used to create new combinations of genetic material, often involving the insertion of a gene into a vector for propagation.

Fluorescent Protein Fusion

A method of studying protein location within cells by attaching a fluorescent protein to a protein of interest.

Genetic Circuits

Biotechnological circuits designed to perform specific functions, like breaking down toxins.

DNA Fingerprinting

Analyzing DNA patterns to identify individuals, including in forensics, genealogy, and parentage testing.

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Diagnostic Electrophoresis

Using electrophoresis to screen for genetic disorders or identify abnormal proteins.

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Polyacrylamide Gel Electrophoresis

A gel matrix used for separating molecules based on size and charge, commonly used for proteins.

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Molecular Sieving

The separation of molecules in a gel based on their size, allowing for the identification and analysis of different components.

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PAGE (Polyacrylamide Gel Electrophoresis)

A technique used to separate components based on charge and size, allowing for the identification and analysis of proteins.

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Electrophoresis

The movement of charged particles like DNA, RNA, or proteins through a solution under the influence of an electric field

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Factors affecting Electrophoretic Mobility

The rate at which a charged particle travels during electrophoresis is influenced by factors like its net charge, size, and the electric field strength.

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Effect of pH on Electrophoretic Mobility

The pH of the solution affects the overall charge of a molecule. A molecule at its zwitterion state (equal positive and negative charges) has zero mobility.

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Zone Electrophoresis

A type of electrophoresis where separated components are distributed into distinct zones on a supporting medium like paper or gel.

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Gel Electrophoresis

A technique used to separate molecules based on their different electrophoretic mobilities

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Moving Boundary Electrophoresis

Electrophoresis techniques where the boundary between zones is continuously moving. Includes capillary electrophoresis, isotachophoresis, and isoelectric focusing.

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Capillary Electrophoresis

A technique used to separate molecules based on their differences in electric charges

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Isoelectric Focusing

A type of electrophoresis where molecules are separated based on their isoelectric points, the pH at which they have no net charge.

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Chromatography

A technique for separating mixtures of unreacted contents into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

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Chromatogram

The visual output of the chromatograph.

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Stationary Phase

The chromatographic packing material held inside the column hardware, needed to effect the separation.

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Mobile Phase

The phase that moves in a definite direction, carrying components through the stationary phase.

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Isoelectric Point (pI)

The pH at which a protein has no net charge.

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Protein Migration in Isoelectric Focusing

A protein will stop migrating in a pH gradient when it reaches a region where its net charge is zero (i.e., its pI).

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High Performance Liquid Chromatography (HPLC)

A technique using a high-pressure pump to force a liquid mobile phase through a stationary phase, separating components based on their affinities for the two phases.

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Fluidics system

Responsible for moving the sample from the sample tube to the flow cell; sorts or transports sample to waste.

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Optics system

Includes excitation light sources, lenses, filters, and detectors. It's responsible for collecting and moving light around the instrument and generating photocurrent.

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Electronics system

The "brain" of the flow cytometer; digitizes and processes the photocurrent from the detector for analysis.

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Forward scatter (FSC)

This measurement detects the cell size or volume.

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Side scatter (SSC)

This measurement reflects the internal complexity of a cell, such as its cytoplasmic content or nuclear structure.

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Flow cytometry

The process of analyzing cells as they pass through a laser beam.

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Laser excitation

A beam of laser light is directed at a stream of fluid containing cells.

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Fluorescence emission

Cells emit fluorescent light at a longer wavelength when excited by the laser.

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HPLC Pump

A pump in HPLC that can generate pressures up to 4000 psi and flow rates up to 10 ml/min.

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Isocratic vs. Gradient Elution

The ability of an HPLC pump to deliver a constant mobile phase composition or to vary the composition over time.

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Sample Injector

A component in HPLC that introduces the sample into the flowing mobile phase. It can be automated (auto-sampler) or operated manually.

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HPLC Column

The heart of the HPLC system, typically made of polished stainless steel, holding a stationary phase.

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Column Internal Diameter (ID)

The internal diameter (ID) of an HPLC column affects the amount of sample that can be injected and the sensitivity of the analysis. Larger columns are typically used for purification, while smaller columns are ideal for high sensitivity.

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Particle Size

The size of particles in the stationary phase. Smaller particles offer greater surface area and better separations. Larger particles provide better flow rates, especially for larger analytes.

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Pore Size

The size of pores within the stationary phase particles. Small pores offer greater surface area, while larger pores are better for larger molecules.

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Direct ELISA

An ELISA method where the antibody is coated on the plate and the antigen is detected by an antibody conjugated to an enzyme.

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Indirect ELISA

An ELISA method where the antigen is coated on the plate and the antibody is detected using a secondary antibody conjugated to an enzyme.

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Competitive ELISA

An ELISA type that measures the amount of antibody in a sample by using a known antigen coated on the plate and various concentrations of the antibody. The higher the antibody concentration, the lower the color intensity.

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Sandwich ELISA

An ELISA type that uses a known antibody coated on the plate and different concentrations of the antigen in the sample. The higher the antigen concentration, the higher the color intensity.

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Sandwich ELISA - Screening Antigen

A method of screening antigens by using ELISA. The antibody is coated on the wells of a microtiter plate, and the antigen is added to the wells. The antigen binds to the antibody, and the bound antigen is detected by an enzyme-linked antibody.

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Competitive ELISA - Screening Antibody

A method of screening antibodies by using ELISA. The antigen is coated on the wells of a microtiter plate, and a mixture of the antigen and antibody is added to the wells. The antibody competes with the enzyme-linked antibody for binding to the antigen, and the amount of bound enzyme-linked antibody is inversely proportional to the amount of antibody in the sample.

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Blocking in ELISA

A key step in ELISA, where a blocking solution is used to prevent false positive results by binding to any remaining unoccupied sites on the plate.

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Color Intensity vs. Quantity in ELISA

In ELISA, the intensity of the color produced is directly proportional to the amount of bound enzyme-linked antibody, which is further correlated to the amount of antibody or antigen present in the sample.

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Study Notes

Practical Part of Spectral Analysis and Applied Spectroscopy (CP508)

  • Course for Biochemistry Diploma Students
  • First Semester, 2024-2025

Biochemical Lab Instruments

  • Contents:
    • Electrophoresis Techniques
    • HPLC
    • Spectrophotometer
    • ELISA
    • Flow Cytometry

Electrophoresis Techniques

  • Electrophoresis is the migration of charged particles (DNA, RNA, proteins) in a solution under an external electric field.
  • Rate of ion migration is influenced by:
    • Net charge of the molecule
    • Size and shape of the particle
    • Strength of the electric field
    • Properties of the supporting medium
    • Temperature
  • Particle mobility depends on particle size and shape, and the medium
  • Different types include:
    • Zone electrophoresis (paper, gel, thin layer, cellulose acetate)
    • Moving boundary electrophoresis (capillary electrophoresis, isotachophoresis, isoelectric focusing)

Paper Electrophoresis

  • Filter paper (Whatman No. 1 and 3) is used in strips (3mm or 5cm wide).
  • Separation takes 12-14 hours.
  • Advantages: economical, easy to use.
  • Disadvantages: Certain compounds (proteins, hydrophilic molecules) cannot be resolved due to the adsorptive and ionogenic properties of paper

Gel Electrophoresis

  • Separation based on molecular sieving (molecular size).
  • Gel material is a colloid (mostly water, Agar, Agarose, Polyacrylamide, Starch).
  • Gel is electrically neutral.
  • Different gels suitable for different molecules.
  • Macromolecule separation, allowing smaller molecule migration is possible.

Agar and Agarose Gels

  • Agar is a mixture of polysaccharides (seaweeds).
  • Agarose is a highly purified polysaccharide.
  • Agarose dissolves in boiling liquid and gels upon cooling.
  • Advantages: easy preparation, high resolution, good protein separation.
  • Disadvantages: electroosmosis is high resolution is lower than polyacrylamide.

Polyacrylamide Gel Electrophoresis (PAGE)

  • Polymerization of acrylamide monomers in the presence of methylene-bis-acrylamide (cross-linking).
  • Covalent cross-links forms a more rigid gel.
  • Suitable for smaller molecules.

Capillary Electrophoresis

  • Charged molecules migrate towards opposite poles.
  • Separates molecules based on characteristics.
  • High voltage across buffer filled capillaries.
  • Capillary can be filled with a gel, eliminating electroosmosis.
  • High resolution and sensitivity.
  • Suitable for analysis of mixtures and detection of small differences.

Isotachophoresis

  • Based on the development of a potential gradient.
  • Higher mobility leading electrolyte (e.g. chloride) and lower mobility trailing electrolyte (e.g. glycinate) are used.
  • Sample ionic components stack into discrete zones.
  • High resolution.

Isoelectric Focusing

  • Based on isoelectric point (pI) of proteins.
  • Proteins migrate to the pH gradient based on net charge.
  • pH of the protein equals the pH of the gradient.
  • Separation of proteins in sharp bands.

Starch Gel Electrophoresis

  • Suspension of granular starch, boiled in a buffer to give a clear colloidal suspension.
  • Cools to form a semisolid gel (amylopectin).
  • Advantages: High resolving power, yields especially proteins.
  • Disadvantages: Electro osmotic effect, variation in pore size.

Thin Layer Electrophoresis

  • Studies done in a thin layer of silica or alumina.
  • Advantages: Less time consuming, good resolution.
  • Applications: combined electrophoretic-chromatographic studies (proteins, nucleic acids)

Cellular Acetate Electrophoresis

  • Contains 2-3 acetyl groups/glucose unit; lesser adsorption capacity than paper.
  • Advantages: No tailing of proteins, good for hydrophilic material, sharp bands, wide range of particle sizes, and high voltage.
  • Disadvantages: Expensive, presence of sulphonates/carboxylic residues cause electroosmosis.

High Performance Liquid Chromatography (HPLC)

  • Separates mixtures of components based on differential affinities with mobile and stationary phases.
  • Mixture is placed on the stationary phase and mobile phase passes through.
  • Mobile phase dissolves the components, carrying them through stationary phase.
  • Components separate based on attraction to each phase.
  • Chromatograph: the equipment; Chromatogram: output display
  • Stationary phase: fixed inside column hardware.
  • Mobile phase: moves in a definite direction.
  • Analyte/Sample: the substance to be separated
  • Retention time: characteristic time for sample to pass system (from injection to peak maximum)
  • Eluate: analyte + eluent (from column to detector)
  • Elution: analyte migration through system and detection.
    • Isocratic elution: mobile phase constant
    • Gradient elution: eluent mixture changes over time
  • HPLC Principle: separation based on hydrophobic interactions between the polar eluent, the relatively nonpolar analyte, and the nonpolar stationary phase

Spectrophotometer

  • Measures intensity of electromagnetic energy at various wavelengths.
  • UV-visible-NIR spectrophotometers operate in UV, visible, and near infrared regions.
  • Light source, monochromator (separates light), and detector.
  • Non-destructive technique.
  • Based on Beer-Lambert law showing relationship between absorbance/amount of absorbed light, path length, and concentration
  • Factors influencing absorbance:
    • sample thickness (path length)
    • concentration
    • chemical nature of the compound

ELISA (Enzyme-linked immunosorbent assay)

  • Biochemical technique detecting/quantifying proteins/antibodies/enzymes/hormones
  • Principle: antigen-antibody interaction
  • Components:
    • Antigen: substance stimulating immune response
    • Antibody: protein made in response to antigen
    • Enzyme Conjugate: antibody joined with enzyme (e.g., HRP, ALP)
    • Substrate: compound converted by enzyme for color change
  • Types: Direct, Indirect, Sandwich, and Competitive.
  • Advantages/Disadvantages vary depending on the specific type.

Flow Cytometry

  • Passes cells/particles in single file through laser beam.
  • Uses fluorescent labeling.
  • Detects cell components/properties based on light scattered/emitted.
  • Measures cell health; identifies and characterizes distinct cell subsets.
  • Uses fluidics, optics and electronics.
  • Analyzes sample with a laser and detectors.
  • Generates graphs showing cell characteristics.

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Practical Part of Spectral Analysis and Applied Spectroscopy (CP508) 2024-2025 PDF

Description

This quiz covers the practical aspects of spectral analysis and applied spectroscopy for biochemistry diploma students. Focus areas include electrophoresis techniques and several biochemical lab instruments like HPLC and spectrophotometers. Dive into the fundamental principles that govern these techniques.

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