Liquid Chromatography and Electrophoresis

Liquid Chromatography and Electrophoresis: Advanced Separation Techniques

Liquid Chromatography (LC) and Electrophoresis are two cornerstone separation techniques in analytical chemistry, indispensable for analyzing complex mixtures in diverse fields such as pharmaceuticals, biotechnology, environmental science, and clinical diagnostics. While both aim to separate components, they achieve this through distinct physical principles: LC relies on differential partitioning between a stationary and mobile phase, whereas electrophoresis utilizes the differential migration of charged species in an electric field.

1. Liquid Chromatography (LC): Principles and Techniques

Liquid Chromatography is a form of chromatography where the mobile phase is a liquid. It is particularly powerful for separating non-volatile and thermally unstable compounds. The most common and advanced form is High-Performance Liquid Chromatography (HPLC).

1.1. HPLC: High-Performance Liquid Chromatography

HPLC evolved from traditional column chromatography by using smaller stationary phase particles and high pressure to force the mobile phase through the column. This results in significantly improved efficiency, speed, and resolution.

1.1.1. HPLC Instrumentation

A typical HPLC system consists of:

  • Solvent Reservoir and Pump: Delivers the mobile phase (often a mixture of solvents) at a precise and constant flow rate (e.g., in mL/min) and high pressure (up to 6000 psi or more).
  • Injector (Autosampler): Introduces a precise volume of sample (e.g., 1-100 µL) into the high-pressure mobile phase stream. Autosamplers allow for automated analysis of multiple samples.
  • Column: The heart of the separation. It’s a stainless steel tube (typically 50-300 mm long, 2.1-4.6 mm ID) packed with small, uniform stationary phase particles (e.g., 1.7-5 µm diameter). The column is often housed in a temperature-controlled oven to maintain consistent conditions.
  • Detector: Senses the separated analytes as they elute from the column. Converts the analyte’s presence into an electrical signal. Common HPLC detectors include:
    • UV-Vis Detector: Most common. Measures absorbance of UV or visible light by analytes. Sensitive for compounds with chromophores.
    • Photodiode Array (PDA) Detector / Diode Array Detector (DAD): A type of UV-Vis detector that collects a full UV-Vis spectrum for each eluting compound, aiding in peak identification and purity assessment.
    • Refractive Index (RI) Detector: Measures changes in the refractive index of the mobile phase as analytes elute. Universal detector (responds to almost any compound) but less sensitive and incompatible with gradient elution.
    • Fluorescence Detector: Highly sensitive and selective for fluorescent compounds.
    • Mass Spectrometer (MS) Detector: Provides molecular weight and structural information, often considered the most powerful LC detector due to its ability to identify compounds definitively.
  • Data Acquisition and Processing System: A computer with software to record detector signals, process data, generate chromatograms, and perform quantitative analysis.

1.1.2. HPLC Elution Modes

  • Isocratic Elution: The mobile phase composition remains constant throughout the entire chromatographic run. Simple and provides stable baselines.
  • Gradient Elution: The mobile phase composition is changed continuously or in steps during the chromatographic run (e.g., increasing the percentage of organic solvent). This is essential for separating complex mixtures with a wide range of polarities, as it allows strongly retained compounds to elute more quickly, improving resolution and reducing run time.

1.1.3. Modes of HPLC Separation (based on stationary phase chemistry)

  1. Reversed-Phase HPLC (RP-HPLC):
    • Mechanism: Based on hydrophobic interactions.
    • Stationary Phase: Non-polar (hydrophobic), most commonly silica particles bonded with C18 (octadecylsilane) or C8 (octylsilane) chains.
    • Mobile Phase: Polar solvent mixture (e.g., water/methanol, water/acetonitrile). Less polar analytes are retained longer.
    • Dominance: The most widely used HPLC mode (over 80% of all HPLC applications).
    • Analytes: Used for a vast range of analytes, from small organic molecules to peptides.
  2. Normal-Phase HPLC (NP-HPLC):
    • Mechanism: Based on polar interactions (hydrogen bonding, dipole-dipole).
    • Stationary Phase: Polar (hydrophilic), e.g., bare silica, alumina.
    • Mobile Phase: Non-polar solvent mixture (e.g., hexane/ethyl acetate). More polar analytes are retained longer.
    • Applications: Used for highly polar, water-insoluble compounds; often sensitive to trace water in the mobile phase.
  3. Ion-Exchange Chromatography (IEC):
    • Mechanism: Separation based on reversible electrostatic interactions between charged analytes (ions) and oppositely charged functional groups covalently bound to the stationary phase resin.
    • Stationary Phase: Contains fixed charged groups (e.g., strong cation exchangers like SO3−​, weak anion exchangers like NH2​).
    • Mobile Phase: Aqueous buffer solution. Separation is controlled by pH and ionic strength.
    • Applications: Separation of ions, amino acids, proteins, nucleotides.
  4. Size-Exclusion Chromatography (SEC) / Gel Permeation Chromatography (GPC) / Gel Filtration Chromatography (GFC):
    • Mechanism: Separation based purely on molecular size (hydrodynamic volume).
    • Stationary Phase: Porous polymer beads (e.g., cross-linked dextran or polyacrylamide).
    • Elution Order: Larger molecules are excluded from the pores and elute first. Smaller molecules penetrate the pores and are retained longer.
    • Applications: Polymer characterization (molecular weight distribution), protein purification, separation of macromolecules.
  5. Affinity Chromatography:
    • Mechanism: Highly specific and reversible biological interactions between the analyte and a specific ligand covalently immobilized on the stationary phase.
    • Stationary Phase: Contains immobilized ligands (e.g., antibody, enzyme, receptor).
    • Applications: Highly selective purification of specific biomolecules (e.g., purifying a specific protein using an antibody against it).

2. Electrophoresis: Principles and Techniques

Electrophoresis is a separation technique that separates charged molecules based on their differential migration rates in an electric field. It is particularly effective for large biomolecules like proteins and nucleic acids.

2.1. Fundamental Principle

  • When a charged molecule is placed in an electric field, it experiences an electrostatic force and migrates towards the electrode of opposite charge.
  • The rate of migration (v) depends on:
    • Net charge (q): Higher charge, faster migration.
    • Friction (or Drag) Force: Depends on the molecule’s size and shape, and the viscosity of the medium.
    • Electric Field Strength (E): Stronger field, faster migration.
  • Electrophoretic Mobility (μep​): A characteristic property of a molecule in a given medium, representing its migration velocity per unit electric field strength.
    • v=μep​E
    • μep​=fq​ (where f is the frictional coefficient)

2.2. Gel Electrophoresis

Most commonly performed in a gel matrix, which acts as a molecular sieve, further separating molecules based on size in addition to charge-to-mass ratio.

2.2.1. Agarose Gel Electrophoresis

  • Medium: Agarose gel (a polysaccharide polymer).
  • Pore Size: Large pores, making it suitable for separating very large molecules like DNA, RNA, and large proteins.
  • Applications: DNA fingerprinting, DNA sequencing, plasmid analysis, RFLP analysis. Nucleic acids are negatively charged due to their phosphate backbone, so they migrate towards the positive electrode.

2.2.2. Polyacrylamide Gel Electrophoresis (PAGE)

  • Medium: Polyacrylamide gel (a synthetic polymer).
  • Pore Size: Can be precisely controlled by adjusting monomer concentration, allowing for separation of smaller molecules with very high resolution.
  • Applications: Primarily used for separating proteins and small nucleic acids.

2.2.3. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

  • Principle: Separates proteins primarily by molecular weight.
  • SDS Role: Sodium Dodecyl Sulfate (SDS) is an anionic detergent that denatures proteins (unfolds them into linear chains) and coats them uniformly, imparting a large, uniform negative charge proportional to their length. This effectively masks the protein’s native charge.
  • Migration: All SDS-coated proteins then migrate towards the positive electrode, with their migration speed determined almost exclusively by their size (molecular weight) through the sieving effect of the polyacrylamide gel. Smaller proteins move faster.
  • Applications: Protein molecular weight determination, protein purity assessment, protein expression analysis.

2.2.4. Isoelectric Focusing (IEF)

  • Principle: Separates proteins based on their isoelectric point (pI), the pH at which a molecule has no net electric charge.
  • Mechanism: Proteins migrate in a pH gradient until they reach the pH equal to their pI, where their net charge is zero, and thus their electrophoretic mobility becomes zero.
  • Applications: High-resolution protein separation, protein characterization, proteomics.

2.3. Capillary Electrophoresis (CE)

A relatively modern technique that performs electrophoretic separations within narrow-bore (10-100 µm internal diameter) fused-silica capillaries. It offers extremely high efficiency and speed.

2.3.1. Key Principles of CE

  • High Electric Field: Very high voltages (up to 30 kV) are applied across the capillary, leading to very strong electric fields.
  • Electroosmotic Flow (EOF): This is a bulk flow of the entire solution within the capillary. It arises from the interaction of the electric field with the charges on the inner surface of the capillary (silanol groups on silica become negatively charged above pH 2-3). Counterions from the buffer form an electrical double layer, and when the electric field is applied, these counterions migrate, dragging the bulk solution with them.
    • EOF is typically directed towards the negative electrode (if the capillary surface is negatively charged, common at neutral/basic pH).
    • Crucially, EOF is essentially flat (plug-like flow), unlike the parabolic flow in pressure-driven chromatography, which significantly reduces band broadening and contributes to the high efficiency of CE.
  • Separation Order (with EOF): If EOF is stronger than electrophoretic migration, both cations, neutral molecules, and anions will all migrate towards the same detector. Cations elute first (strongest migration + EOF), followed by neutral molecules (only carried by EOF), and then anions (electrophoretic migration opposes EOF, so they are slowest).

2.3.2. Modes of CE

  1. Capillary Zone Electrophoresis (CZE):
    • Simplest and most common CE mode.
    • Separates analytes based purely on their charge-to-mass ratio and hydrodynamic radius in a free solution (no gel matrix).
    • Operates with a uniform buffer throughout the capillary.
  2. Capillary Gel Electrophoresis (CGE):
    • Similar to SDS-PAGE but performed in a capillary filled with a polymer network (gel).
    • Separates molecules primarily by size, typically used for DNA sequencing fragments and SDS-protein complexes.
  3. Capillary Isoelectric Focusing (cIEF):
    • Performs IEF in a capillary.
    • Separates proteins based on their pI.
  4. Micellar Electrokinetic Chromatography (MEKC):
    • A hybrid technique where a surfactant (like SDS) is added to the buffer at concentrations above its critical micelle concentration.
    • Neutral molecules can partition into these micelles, which have a different electrophoretic mobility than the bulk solution. This allows for the separation of neutral compounds.

2.3.3. CE Instrumentation

  • High-Voltage Power Supply: Generates up to 30 kV.
  • Capillary: Fused-silica capillary.
  • Inlet and Outlet Buffer Vials: Contain the electrolyte buffer.
  • Electrodes: Connect the power supply to the buffer vials.
  • Detector: Typically on-column (UV-Vis, PDA, Fluorescence, MS) due to the small sample volumes. The detector window is often created by burning off a small section of the polyimide coating on the capillary.
  • Autosampler: For automated sample introduction.

2.4. Detection in Electrophoresis

  • UV-Vis and Fluorescence: Common for detecting nucleic acids (at 260 nm) and proteins (at 280 nm) or fluorescently labeled compounds.
  • Staining (for Gels): Gels are often stained after separation (e.g., ethidium bromide for DNA, Coomassie Blue or silver stain for proteins) to visualize the separated bands.
  • Mass Spectrometry (MS): Online coupling of CE with MS is increasingly common, providing highly sensitive detection and definitive identification of separated components.

Conclusion

Liquid Chromatography and Electrophoresis are highly complementary separation techniques, each offering unique advantages for different types of analytical challenges. HPLC is widely used for a broad range of molecules, from small pharmaceuticals to large biopolymers, leveraging diverse interaction mechanisms. Electrophoresis, particularly valuable for charged macromolecules like DNA and proteins, offers exceptional resolution based on charge and size, especially evident in advanced techniques like SDS-PAGE and capillary electrophoresis. Together, these methods form the backbone of modern analytical and bioanalytical laboratories, enabling unprecedented insights into the composition and properties of complex chemical and biological systems.

Liquid Chromatography and Electrophoresis: Multiple Choice Questions

Instructions: Choose the best answer for each question. Explanations are provided after each question.

1. In High-Performance Liquid Chromatography (HPLC), what is the typical state of the stationary phase? a) A gas b) A liquid coated on a solid support or a solid particulate c) A supercritical fluid d) The sample itself e) The mobile phase

Explanation: HPLC columns are packed with small solid particles, which may be bare solids or have a liquid phase chemically bonded to their surface.

2. Which component of an HPLC system is responsible for delivering the mobile phase at a precise and constant flow rate under high pressure? a) Injector b) Column c) Detector d) Pump e) Data system

Explanation: The pump is the driving force that propels the mobile phase through the HPLC system.

3. What is the most common mode of HPLC, utilizing a non-polar stationary phase and a polar mobile phase? a) Normal-Phase HPLC b) Ion-Exchange Chromatography c) Size-Exclusion Chromatography d) Reversed-Phase HPLC e) Affinity Chromatography

Explanation: Reversed-phase HPLC is the most prevalent mode, separating compounds based on their hydrophobicity.

4. What is the main advantage of using gradient elution in HPLC compared to isocratic elution? a) Simpler operation. b) Faster analysis for all compounds. c) Better separation of complex mixtures with a wide range of polarities. d) Improved detector sensitivity. e) Reduced solvent consumption.

Explanation: Gradient elution allows for optimal separation of compounds that have very different retention characteristics, by changing the mobile phase strength during the run.

5. In electrophoresis, what is the primary factor that causes charged molecules to migrate? a) Gravity b) Differential solubility c) An electric field d) Molecular size e) Temperature gradient

Explanation: Electrophoresis uses an applied electric field to induce movement of charged species.

6. Which term describes the migration velocity of a charged molecule per unit electric field strength in electrophoresis? a) Retention factor b) Electrophoretic mobility c) Diffusion coefficient d) Viscosity e) Conductivity

Explanation: Electrophoretic mobility is a measure of how fast a charged molecule moves in a given electric field, and it’s characteristic of the molecule and the medium.

7. SDS-PAGE is primarily used to separate proteins based on which property? a) Isoelectric point (pI) b) Native three-dimensional structure c) Molecular weight d) Overall charge e) Enzyme activity

Explanation: SDS denatures proteins and coats them with a uniform negative charge, so their migration through the polyacrylamide gel is almost exclusively determined by their size (molecular weight).

8. What is the role of SDS (Sodium Dodecyl Sulfate) in SDS-PAGE? a) To stain the proteins for visualization. b) To make the gel rigid. c) To denature proteins and impart a uniform negative charge. d) To act as a buffer. e) To increase the viscosity of the solution.

Explanation: SDS ensures that all proteins migrate based solely on their size, overcoming differences in native charge and shape.

9. In Capillary Electrophoresis (CE), what is “electroosmotic flow (EOF)”? a) The flow of analytes due to their charge. b) The bulk flow of the entire solution within the capillary, caused by the electric field acting on charges at the capillary wall. c) The diffusion of analytes from high to low concentration. d) The flow of mobile phase due to pressure. e) The movement of ions in the buffer.

Explanation: EOF is a distinct phenomenon in CE where the entire buffer solution moves due to the electric field’s interaction with the charged capillary surface.

10. Which type of detector in HPLC provides a full UV-Vis spectrum for each eluting compound, aiding in peak identification? a) Refractive Index Detector (RID) b) Fluorescence Detector c) Mass Spectrometer (MS) d) Photodiode Array (PDA) Detector e) Electrochemical Detector

Explanation: PDA detectors collect absorbance data across a range of wavelengths simultaneously, providing spectral information for each peak.

11. Which HPLC mode is best suited for separating high molecular weight polymers and proteins based solely on their size? a) Reversed-Phase HPLC b) Normal-Phase HPLC c) Ion-Exchange Chromatography d) Size-Exclusion Chromatography (SEC) e) Affinity Chromatography

Explanation: SEC (also known as GPC or GFC) is specifically designed to separate molecules based on their hydrodynamic volume as they pass through pores in the stationary phase.

12. In which type of chromatography does the stationary phase contain immobilized ligands for specific biological interactions? a) Partition Chromatography b) Adsorption Chromatography c) Ion-Exchange Chromatography d) Size-Exclusion Chromatography e) Affinity Chromatography

Explanation: Affinity chromatography uses highly specific binding interactions (like antibody-antigen or enzyme-substrate) for purification.

13. Agarose gel electrophoresis is most commonly used for the separation of: a) Small organic molecules b) DNA and RNA c) Small peptides d) Ions e) Sugars

Explanation: Agarose gels have large pores, making them ideal for separating large nucleic acid molecules.

14. What is the primary advantage of Capillary Electrophoresis (CE) over traditional gel electrophoresis? a) Lower voltage requirements. b) Slower separation times. c) Extremely high efficiency and speed. d) Less sensitive detection. e) Requires larger sample volumes.

Explanation: CE uses narrow capillaries and high electric fields to achieve very rapid and highly efficient separations with narrow peaks.

15. What type of HPLC elution involves a constant mobile phase composition throughout the run? a) Gradient elution b) Isocratic elution c) Isothermal elution d) Isoelectric elution e) Stepwise elution

Explanation: Isocratic elution uses a single, unchanging mobile phase composition.

16. Which HPLC detector is considered “universal” (responds to almost any compound) but has lower sensitivity and cannot be used with gradient elution? a) UV-Vis Detector b) Fluorescence Detector c) Mass Spectrometer (MS) d) Refractive Index (RI) Detector e) Electrochemical Detector

Explanation: RI detectors measure changes in the bulk property of the mobile phase, making them universal but highly sensitive to changes in mobile phase composition, hence incompatible with gradients.

17. If proteins are separated by Isoelectric Focusing (IEF), what property are they separated by? a) Molecular weight b) Hydrophobicity c) Net charge d) Isoelectric point (pI) e) Shape

Explanation: IEF separates proteins based on their pI, where they migrate in a pH gradient until they reach a point where their net charge is zero.

18. What is the charge of DNA molecules at neutral pH, causing them to migrate towards the positive electrode in electrophoresis? a) Positive b) Neutral c) Negative d) Variable depending on sequence e) Zero

Explanation: The phosphate backbone of DNA (and RNA) carries a negative charge at neutral pH, making these molecules migrate towards the anode.

19. Which HPLC mode utilizes a polar stationary phase (e.g., bare silica) and a non-polar mobile phase? a) Reversed-Phase HPLC b) Normal-Phase HPLC c) Ion-Exchange Chromatography d) Size-Exclusion Chromatography e) Affinity Chromatography

Explanation: Normal-phase HPLC relies on the interaction between polar analytes and a polar stationary phase.

20. What is a key advantage of directly coupling Mass Spectrometry (MS) to HPLC or CE? a) It increases the separation efficiency. b) It reduces sample preparation time. c) It provides definitive molecular weight and structural identification of separated components. d) It is a cheaper detection method. e) It eliminates the need for a column.

Explanation: MS provides highly specific and detailed information about the chemical identity of the eluted analytes, making it a powerful confirmation tool.

21. In CE, if the electroosmotic flow (EOF) is stronger than the electrophoretic migration, how will all charged species and neutral molecules eventually move towards the detector? a) Only cations will move. b) Only anions will move. c) All species will move towards the anode (positive electrode) if EOF is towards the anode. d) All species will move towards the cathode (negative electrode). e) They will not move at all.

Explanation: If EOF is strong enough and directed towards the detector (e.g., anode at typical pH), it will carry all species, including neutral molecules and even anions migrating in the opposite direction, towards the detector.

22. Which technique is commonly used to separate proteins based on their size after being denatured and uniformly charged? a) Agarose gel electrophoresis b) Capillary Zone Electrophoresis (CZE) c) SDS-PAGE d) Isoelectric Focusing (IEF) e) Ion-Exchange Chromatography

Explanation: SDS-PAGE specifically utilizes SDS to prepare proteins for separation primarily by molecular weight.

23. What is the function of the column oven in an HPLC system? a) To vaporize the sample. b) To cool the mobile phase. c) To maintain a constant and optimal temperature for consistent separation. d) To dry the column. e) To illuminate the column for detection.

Explanation: Maintaining a constant column temperature is crucial for reproducible retention times and peak shapes in HPLC.

24. Which type of HPLC detector measures the change in refractive index of the eluent, making it useful for compounds without chromophores? a) UV-Vis Detector b) Fluorescence Detector c) Mass Spectrometer (MS) d) Photodiode Array (PDA) Detector e) Refractive Index (RI) Detector

Explanation: The RI detector is a bulk property detector, sensing differences in the mobile phase’s refractive index caused by the presence of analytes.

25. If an analyte is very polar and you are using Normal-Phase HPLC, how would its retention factor (k) likely be? a) Very small (elutes quickly). b) Very large (retained strongly). c) Zero. d) Negative. e) Independent of polarity.

Explanation: In normal-phase HPLC, the polar stationary phase strongly interacts with polar analytes, leading to high retention.

26. Why are narrow-bore capillaries used in Capillary Electrophoresis (CE)? a) To increase resistance to flow. b) To reduce sample consumption. c) To dissipate heat efficiently and reduce band broadening. d) To make the instrument smaller. e) To increase detector sensitivity.

Explanation: Small diameters allow for efficient heat dissipation, preventing thermal broadening and enabling the use of high electric fields for high efficiency.

27. What happens to proteins during SDS-PAGE sample preparation? a) They fold into their native 3D structure. b) They are fragmented into smaller peptides. c) They are denatured and coated with a uniform negative charge. d) They are specifically labeled with a fluorescent tag. e) They are complexed with metal ions.

Explanation: SDS denatures the proteins, linearizes them, and imparts a negative charge proportional to their size.

28. Which of the following best describes isocratic elution in HPLC? a) Mobile phase composition changes throughout the run. b) Mobile phase flow rate changes throughout the run. c) Mobile phase composition remains constant throughout the run. d) Temperature changes throughout the run. e) Sample concentration changes throughout the run.

Explanation: Isocratic means “constant strength” in reference to the mobile phase composition.

29. What is the fundamental difference in the separation principle between HPLC and electrophoresis? a) HPLC separates by charge, electrophoresis by size. b) HPLC separates by polarity, electrophoresis by mass. c) HPLC uses differential partitioning, electrophoresis uses differential migration in an electric field. d) HPLC is for volatile compounds, electrophoresis for non-volatile. e) HPLC uses gas mobile phase, electrophoresis uses liquid.

Explanation: HPLC separates based on how analytes distribute between two phases, while electrophoresis separates based on their movement in an electric field.

30. Which type of chromatography is highly selective and used to purify specific biomolecules like antibodies or enzymes? a) Size-Exclusion Chromatography b) Ion-Exchange Chromatography c) Adsorption Chromatography d) Affinity Chromatography e) Partition Chromatography

Explanation: Affinity chromatography uses specific biochemical interactions to isolate target molecules.

31. In a CE run, if cations, neutral molecules, and anions are present, and the EOF is directed towards the detector (anode), what will be the typical elution order? a) Anions, Neutrals, Cations b) Cations, Neutrals, Anions c) Neutrals, Anions, Cations d) All elute at the same time e) Anions and Cations only

Explanation: Cations move fastest towards the anode (electrophoretic migration + EOF). Neutrals only move with EOF. Anions move slowest or even migrate away from the detector (electrophoretic migration opposes EOF), so they are last to reach the anode detector if at all.

32. What is a common application of ion-exchange chromatography? a) Separating fat-soluble vitamins. b) Determining molecular weight distribution of polymers. c) Separating amino acids and proteins based on charge. d) Analyzing volatile organic compounds. e) Purifying DNA fragments by size.

Explanation: IEC is specifically designed for the separation of charged species like ions, amino acids, and proteins, based on their net charge.

33. What is the purpose of “staining” in gel electrophoresis? a) To denature the molecules. b) To make the separated bands visible. c) To apply the electric field. d) To load the sample into the gel. e) To control the pH.

Explanation: Most biomolecules (DNA, proteins) are colorless, so staining (e.g., ethidium bromide for DNA, Coomassie Blue for protein) is necessary to visualize them after separation.

34. Which HPLC detector measures the change in absorbance at a single or multiple fixed wavelengths? a) Fluorescence Detector b) Mass Spectrometer (MS) c) Refractive Index Detector (RID) d) UV-Vis Detector e) Electrochemical Detector

Explanation: UV-Vis detectors are standard for compounds with chromophores, measuring light absorption at specific wavelengths.

35. What is the main reason for band broadening in chromatography due to “eddy diffusion”? a) Diffusion of analytes against the flow. b) Slow equilibrium between stationary and mobile phases. c) Different path lengths taken by analytes through the packed column particles. d) Temperature fluctuations in the column. e) Too high mobile phase flow rate.

Explanation: Eddy diffusion (A term in Van Deemter) arises because solute molecules can take different, uneven paths through the irregular packing of the stationary phase.

36. For protein separation, if you need to maintain the native structure and activity of the protein, which technique would be most suitable? a) SDS-PAGE b) Denaturing PAGE c) Size-Exclusion Chromatography (SEC) d) Isoelectric Focusing (IEF) e) MEKC

Explanation: SEC (Gel Filtration) separates proteins based on size in their native conformation, without denaturing agents, making it suitable for maintaining activity. SDS-PAGE and denaturing PAGE denature proteins.

37. Which material is commonly used for the capillary in Capillary Electrophoresis? a) Glass b) Stainless steel c) Fused silica d) Plastic e) Copper

Explanation: Fused silica capillaries are favored in CE due to their excellent optical properties, precise dimensions, and the ability to generate stable electroosmotic flow.

38. What is a “chromatogram”? a) A diagram of the HPLC instrument. b) A visual representation of a molecule’s structure. c) A graph showing detector response versus time or volume of mobile phase. d) A table of chemical properties. e) A map of the separation pathway.

Explanation: The chromatogram is the final output of a chromatographic analysis, displaying peaks for separated components.

39. In HPLC, what is a potential disadvantage of using smaller stationary phase particles (e.g., 1.7 µm vs. 5 µm)? a) Lower column efficiency. b) Increased backpressure, requiring higher pump pressure. c) Slower analysis times. d) Reduced resolution. e) Less flexible mobile phase options.

Explanation: Smaller particles lead to significantly higher backpressure, which requires more robust pumps but in turn results in higher efficiency and better resolution.

40. Which electrophoresis technique is used to separate neutral molecules by incorporating them into micelles? a) Agarose gel electrophoresis b) SDS-PAGE c) Capillary Zone Electrophoresis (CZE) d) Micellar Electrokinetic Chromatography (MEKC) e) Isoelectric Focusing (IEF)

Explanation: MEKC is a specialized CE technique that uses micelles to separate neutral compounds, which would otherwise not separate in standard CZE.

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