Advanced Biochemical Techniques: Chromatography, Spectroscopy and Electrophoresis – Principles and Applications

Introduction

Biochemical techniques play a crucial role in research, diagnostics, and industrial applications. Three fundamental techniques—Chromatography, Spectroscopy, and Electrophoresis—are widely used for molecular separation, identification, and analysis. This study module explores the principles, types, and applications of these techniques.

Best chromatography techniques for proteins,
Spectroscopy applications in biochemistry,
Electrophoresis for DNA analysis,
Low-cost lab chromatography methods,
Spectrophotometry vs fluorometry analysis.

1. Chromatography

1.1 Principle of Chromatography

Chromatography is a separation technique where a mixture is passed through a stationary phase while a mobile phase carries the components at different rates. Separation occurs based on molecular interactions like adsorption, partitioning, or size exclusion.

1.2 Types of Chromatography

  • Paper Chromatography: Used for separating pigments and small organic molecules.
  • Thin Layer Chromatography (TLC): Similar to paper chromatography but with a solid stationary phase like silica or alumina.
  • Gas Chromatography (GC): Separates volatile compounds based on their affinity to the stationary phase.
  • High-Performance Liquid Chromatography (HPLC): A powerful technique for separating biomolecules such as proteins, lipids, and pharmaceuticals.
  • Ion Exchange Chromatography: Separates molecules based on charge interactions.
  • Affinity Chromatography: Utilizes specific ligand-receptor interactions for purification, such as antigen-antibody binding.

1.3 Applications of Chromatography

  • Drug testing and pharmaceutical research.
  • Food safety analysis.
  • Environmental monitoring and pollutant detection.
  • Forensic science and toxicology.

2. Spectroscopy

2.1 Principle of Spectroscopy

Spectroscopy is the study of how matter interacts with electromagnetic radiation. Different spectroscopic techniques measure absorbance, emission, or scattering of light to determine molecular composition and structure.

2.2 Types of Spectroscopy

  • UV-Visible Spectroscopy: Measures absorbance of ultraviolet or visible light; commonly used for protein and nucleic acid quantification.
  • Infrared (IR) Spectroscopy: Detects molecular vibrations and functional groups in organic compounds.
  • Nuclear Magnetic Resonance (NMR) Spectroscopy: Analyzes atomic nuclei behavior in a magnetic field to determine molecular structure.
  • Fluorescence Spectroscopy: Detects fluorescence emissions from excited molecules, widely used in biochemical assays.
  • Mass Spectrometry (MS): Determines molecular weight and structure based on mass-to-charge ratios.

2.3 Applications of Spectroscopy

  • Structural elucidation of biomolecules.
  • Identification of unknown compounds.
  • Studying enzyme kinetics and molecular interactions.
  • Drug discovery and pharmacokinetics.

3. Electrophoresis

3.1 Principle of Electrophoresis

Electrophoresis is a technique for separating charged molecules in an electric field. The movement of molecules depends on size, charge, and shape.

3.2 Types of Electrophoresis

  • Agarose Gel Electrophoresis: Used primarily for DNA and RNA separation.
  • Polyacrylamide Gel Electrophoresis (PAGE): Used for protein separation; can be denaturing (SDS-PAGE) or non-denaturing (native PAGE).
  • Capillary Electrophoresis (CE): High-resolution separation of small molecules and ions.
  • Two-Dimensional Gel Electrophoresis (2D-GE): Separates proteins based on isoelectric point and molecular weight.

3.3 Applications of Electrophoresis

  • DNA fingerprinting and genetic testing.
  • Protein analysis in biomedical research.
  • Diagnosis of genetic disorders and infectious diseases.
  • Quality control in biotechnology industries.

Website URLs for Further Understanding

For more in-depth study, visit these authoritative websites:

Conclusion

Chromatography, Spectroscopy, and Electrophoresis are indispensable biochemical techniques for analyzing and characterizing biomolecules. Their applications extend across scientific research, medicine, and industry, making them essential tools for modern biochemistry. Understanding these techniques equips students and researchers with the knowledge to excel in the field of molecular science.

MCQs with Answers on Biochemical Techniques: Chromatography, Spectroscopy and Electrophoresis

Chromatography

  1. Which principle does chromatography primarily rely on?
    a) Molecular weight
    b) Differential partitioning between phases ✅
    c) Electrical conductivity
    d) Magnetic properties

    Explanation: Chromatography separates components based on their differing affinities between a stationary and a mobile phase.

  2. Which of the following is NOT a type of chromatography?
    a) Gas Chromatography (GC)
    b) High-Performance Liquid Chromatography (HPLC)
    c) Electrophoresis ✅
    d) Thin Layer Chromatography (TLC)

    Explanation: Electrophoresis is a technique used to separate charged molecules in an electric field, not a chromatographic method.

  3. In Thin Layer Chromatography (TLC), the stationary phase is usually made of:
    a) Silica gel or alumina ✅
    b) Agarose
    c) Cellulose
    d) Polyacrylamide

    Explanation: Silica gel or alumina is commonly used for TLC because of their high adsorption properties.

  4. What is the primary application of gas chromatography?
    a) Separation of volatile compounds ✅
    b) Identification of amino acids
    c) DNA sequencing
    d) Protein purification

    Explanation: Gas chromatography is ideal for analyzing volatile and thermally stable compounds.

  5. Which detector is commonly used in High-Performance Liquid Chromatography (HPLC)?
    a) Flame Ionization Detector (FID)
    b) UV-Vis Detector ✅
    c) Thermal Conductivity Detector (TCD)
    d) Refractive Index Detector (RID)

    Explanation: UV-Vis detectors are widely used in HPLC due to their ability to detect a broad range of compounds.

  6. In size-exclusion chromatography, which molecules elute first?
    a) Smaller molecules
    b) Medium-sized molecules
    c) Larger molecules ✅
    d) Negatively charged molecules

    Explanation: Larger molecules elute first because they cannot enter the pores of the stationary phase and travel faster through the column.

  7. Which chromatography technique is best for separating charged biomolecules?
    a) Ion-exchange chromatography ✅
    b) Affinity chromatography
    c) Paper chromatography
    d) Gas chromatography

    Explanation: Ion-exchange chromatography separates molecules based on charge interactions with a charged stationary phase.

  8. Which of the following is a characteristic of affinity chromatography?
    a) Separation based on molecular weight
    b) Use of a ligand that binds specifically to the target molecule ✅
    c) Use of a volatile mobile phase
    d) Use of an electric field for separation

    Explanation: Affinity chromatography employs a specific ligand that selectively binds to the target molecule, facilitating its separation.

Spectroscopy

  1. What does UV-Vis spectroscopy primarily measure?
    a) Scattering of light
    b) Absorption of light ✅
    c) Reflection of light
    d) Transmission of sound

    Explanation: UV-Vis spectroscopy measures the absorbance of UV or visible light by molecules, indicating their concentration.

  2. Which law governs UV-Vis spectroscopy?
    a) Beer-Lambert Law ✅
    b) Newton’s Law
    c) Charles’ Law
    d) Avogadro’s Law

Explanation: The Beer-Lambert Law states that absorbance is directly proportional to the concentration of the absorbing species.

  1. Infrared (IR) spectroscopy is used to determine:
    a) Molecular weight
    b) Functional groups in a molecule ✅
    c) pH of a solution
    d) Protein structure

Explanation: IR spectroscopy detects functional groups based on characteristic bond vibrations.

  1. Which spectroscopy technique is used to study protein secondary structures?
    a) Mass Spectrometry
    b) Circular Dichroism (CD) Spectroscopy ✅
    c) NMR Spectroscopy
    d) Atomic Absorption Spectroscopy

Explanation: CD spectroscopy helps in analyzing α-helix and β-sheet content in proteins.

  1. Fluorescence spectroscopy relies on:
    a) Emission of light from excited molecules ✅
    b) Absorption of X-rays
    c) Scattering of neutrons
    d) Molecular weight determination

Explanation: Fluorescence occurs when a molecule absorbs light at one wavelength and emits light at a longer wavelength.

  1. Which of the following techniques is best for analyzing isotopes?
    a) NMR Spectroscopy
    b) Mass Spectrometry ✅
    c) UV-Vis Spectroscopy
    d) Raman Spectroscopy

Explanation: Mass spectrometry separates isotopes based on their mass-to-charge ratio (m/z).

  1. Nuclear Magnetic Resonance (NMR) spectroscopy primarily uses which type of radiation?
    a) X-rays
    b) Infrared
    c) Radio waves ✅
    d) Ultraviolet

Explanation: NMR spectroscopy uses radio waves to excite nuclear spins in a magnetic field.

Electrophoresis

  1. Electrophoresis is used to separate molecules based on:
    a) Molecular weight and charge ✅
    b) Absorption spectrum
    c) Solubility in solvents
    d) Hydrophobicity

Explanation: Charged molecules move in an electric field based on their size and charge.

  1. Which gel is commonly used for DNA electrophoresis?
    a) Polyacrylamide
    b) Agarose ✅
    c) Cellulose acetate
    d) Silica gel

Explanation: Agarose gel is suitable for DNA electrophoresis due to its large pore size.

  1. SDS-PAGE separates proteins based on:
    a) Charge
    b) Molecular weight ✅
    c) Hydrophobicity
    d) pH

Explanation: SDS denatures proteins and imparts a uniform negative charge, allowing separation based on size.

  1. The purpose of the buffer in electrophoresis is to:
    a) Conduct electricity ✅
    b) Bind to DNA
    c) Provide a pH gradient
    d) Reduce sample viscosity

Explanation: The buffer provides ions to maintain conductivity and pH stability.

  1. In 2D gel electrophoresis, proteins are separated based on:
    a) Charge and molecular weight ✅
    b) Absorption and emission
    c) Density and viscosity
    d) Boiling and melting points

Explanation: First, proteins are separated by charge (isoelectric focusing), then by molecular weight (SDS-PAGE).

Chromatography

  1. Which of the following is used as a mobile phase in Gas Chromatography (GC)?
    a) Liquid solvent
    b) Helium or nitrogen gas ✅
    c) Aqueous buffer
    d) Organic polymer

Explanation: In GC, an inert carrier gas (like helium or nitrogen) transports the analytes through the column.

  1. Reverse-phase chromatography uses a stationary phase that is:
    a) Hydrophilic
    b) Hydrophobic ✅
    c) Charged
    d) Amphiphilic

Explanation: Reverse-phase chromatography employs a non-polar (hydrophobic) stationary phase and a polar mobile phase.

  1. Which type of chromatography is commonly used for protein purification?
    a) Gas Chromatography
    b) Affinity Chromatography ✅
    c) Thin Layer Chromatography
    d) Paper Chromatography

Explanation: Affinity chromatography utilizes a ligand that selectively binds to the target protein, ensuring effective purification.

  1. What does the Rf value in Thin Layer Chromatography (TLC) indicate?
    a) Ratio of solute to solvent concentration
    b) Ratio of distance traveled by solute to distance traveled by solvent ✅
    c) Absorbance ratio
    d) Retention coefficient

Explanation: The Rf value (Retention Factor) helps in identifying compounds by comparing their movement in the stationary phase.

Spectroscopy

  1. Which of the following spectroscopy techniques is best suited for metal ion analysis?
    a) UV-Vis Spectroscopy
    b) Atomic Absorption Spectroscopy (AAS) ✅
    c) Infrared Spectroscopy
    d) Fluorescence Spectroscopy

Explanation: AAS measures the absorption of light by metal ions in a flame, making it ideal for trace metal analysis.

  1. Which spectroscopic method is commonly used for structural determination of organic compounds?
    a) UV-Vis Spectroscopy
    b) Infrared (IR) Spectroscopy
    c) Nuclear Magnetic Resonance (NMR) Spectroscopy ✅
    d) Atomic Absorption Spectroscopy

Explanation: NMR spectroscopy provides detailed information about the structure of organic molecules based on atomic environments.

  1. The primary function of a monochromator in spectrophotometry is to:
    a) Detect light intensity
    b) Select a specific wavelength of light ✅
    c) Amplify the signal
    d) Measure fluorescence

Explanation: A monochromator isolates a single wavelength of light from a broad spectrum for accurate analysis.

  1. Raman Spectroscopy is based on:
    a) Absorption of ultraviolet light
    b) Scattering of monochromatic light ✅
    c) Emission of radiation
    d) Separation of charged molecules

Explanation: Raman spectroscopy detects molecular vibrations by measuring scattered light after laser excitation.

  1. Which of the following is a limitation of UV-Vis spectroscopy?
    a) Cannot detect conjugated systems
    b) Cannot differentiate between different functional groups ✅
    c) Cannot be used for quantitative analysis
    d) Only works with gases

Explanation: UV-Vis spectroscopy detects absorbance but does not provide specific functional group information like IR spectroscopy.

Electrophoresis

  1. Which factor does NOT affect the migration rate in gel electrophoresis?
    a) Voltage applied
    b) Molecular weight of the molecule
    c) Temperature
    d) Wavelength of light used ✅

Explanation: Electrophoresis depends on electric field strength, molecular weight, and gel composition, but not light wavelength.

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