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B.Sc. 4th Year Bioinstrumentation

Section 2: Electrophoretic Techniques and Chromatography

Detailed examination-oriented notes covering the principles, instrumentation, procedures, interpretation, applications, advantages, limitations and important questions of electrophoresis and chromatographic techniques.

UNIT 5 — ELECTROPHORETIC TECHNIQUES

5.1 Introduction to Electrophoresis

Definition: Electrophoresis is a separation technique in which charged particles or molecules migrate through a medium under the influence of an applied electric field. The rate and direction of migration depend on factors such as net charge, molecular size, shape, electric field strength, buffer composition and properties of the supporting medium.

Electrophoresis is one of the most important analytical and preparative techniques used in biochemistry, molecular biology, microbiology, genetics, biotechnology and biomedical research. It is particularly useful for separating proteins, DNA, RNA and other charged biological molecules.

The basic principle is simple. When a charged molecule is placed in an electric field, it experiences an electrical force and moves toward the electrode carrying the opposite charge. Positively charged molecules move toward the cathode, while negatively charged molecules move toward the anode. However, the actual movement through a gel is influenced not only by electrical charge but also by molecular size and shape.

Principle of electrophoresis
Figure 27. Principle of electrophoresis showing migration of charged biological molecules through a supporting medium.

5.2 Basic Principle of Electrophoretic Migration

When a charged molecule is placed in an electric field, the electrical force acting on the molecule causes migration. The direction of movement depends on the sign of the molecular charge.

The electrophoretic mobility of a molecule is affected by the relationship between electrical driving force and resistance produced by the medium. Small molecules generally pass through a porous gel more easily than large molecules. Therefore, biological molecules can often be separated according to molecular size.

Electrophoretic mobility = Distance migrated / Time

In practical electrophoresis, the migration rate is also affected by buffer pH, ionic strength, gel concentration, voltage, temperature, molecular conformation and the charge-to-mass ratio of the molecule.

5.3 Main Components of an Electrophoresis System

  1. Power supply: Provides controlled direct electrical current or voltage.
  2. Electrophoresis chamber: Contains the gel and running buffer.
  3. Electrodes: Provide the electrical field required for migration.
  4. Supporting medium: Usually agarose or polyacrylamide gel.
  5. Running buffer: Maintains appropriate pH and provides ions to conduct electricity.
  6. Sample loading system: Used to introduce biological samples into wells.
  7. Detection system: Used to visualize separated molecules.

5.4 Factors Affecting Electrophoretic Separation

Important examination point: For nucleic acids separated through agarose gel, molecular size is the major determinant of migration because DNA molecules have a relatively uniform negative charge-to-mass ratio. Smaller DNA fragments generally migrate farther through the gel than larger fragments.

5.5 Agarose Gel Electrophoresis

Agarose gel electrophoresis is primarily used for the separation and analysis of DNA and RNA molecules. Agarose is a polysaccharide obtained from agar and forms a porous three-dimensional network when dissolved in hot buffer and allowed to cool.

Agarose gel electrophoresis
Figure 28. Agarose gel electrophoresis showing wells, DNA migration and separated bands.

Principle

DNA molecules possess negatively charged phosphate groups. When an electric field is applied, DNA migrates toward the positive electrode. The agarose matrix acts as a molecular sieve. Smaller DNA fragments move through the pores more rapidly and therefore travel farther than larger fragments.

Preparation of Agarose Gel

  1. Prepare the required concentration of agarose in an appropriate electrophoresis buffer.
  2. Heat the mixture until the agarose dissolves completely.
  3. Allow the solution to cool sufficiently before adding a suitable nucleic-acid visualization reagent when appropriate.
  4. Place a comb in the casting tray to form wells.
  5. Pour the molten agarose into the casting tray.
  6. Allow the gel to solidify.
  7. Remove the comb carefully.
  8. Place the gel in the electrophoresis chamber.

Sample Loading

DNA samples are mixed with loading dye. The loading dye increases sample density so that the sample sinks into the wells and also provides tracking dyes that help monitor electrophoresis.

Applications of Agarose Gel Electrophoresis

Advantages

Limitations

5.6 Polyacrylamide Gel Electrophoresis — PAGE

Polyacrylamide gel electrophoresis, commonly abbreviated as PAGE, uses a polyacrylamide matrix as the supporting medium. It provides higher resolution than agarose for many protein and small nucleic-acid applications.

Polyacrylamide gel electrophoresis
Figure 29. Polyacrylamide gel electrophoresis system.

Principle

Polyacrylamide forms a finely controlled porous matrix. Molecules migrate through the pores according to their charge, size and shape. The properties of the gel can be modified by changing the concentration and composition of acrylamide and cross-linking components.

Types of PAGE

5.7 SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, commonly called SDS-PAGE, is one of the most widely used techniques for analyzing proteins.

SDS PAGE protein separation
Figure 30. SDS-PAGE showing protein bands separated according to molecular size.

Principle of SDS-PAGE

SDS is an anionic detergent that binds to hydrophobic regions of proteins and disrupts many non-covalent interactions. Together with suitable sample treatment, it causes proteins to become largely unfolded and gives them a relatively similar negative charge-to-mass relationship.

As a result, the major factor determining migration through the polyacrylamide gel becomes molecular size. Smaller proteins generally move more rapidly through the gel than larger proteins.

Common Components

General Procedure

  1. Prepare protein samples.
  2. Mix samples with appropriate loading buffer.
  3. Apply heat when the experimental protocol requires denaturation.
  4. Load samples into gel wells.
  5. Load a molecular-weight marker.
  6. Apply an electrical field.
  7. Allow proteins to migrate through the gel.
  8. Stop electrophoresis when suitable separation has been achieved.
  9. Stain the gel using an appropriate protein staining method.
  10. Destain when required.
  11. Analyze the resulting protein bands.

Applications of SDS-PAGE

Limitations

5.8 Native PAGE

Native PAGE is performed under conditions that attempt to preserve the natural structure and biological properties of proteins. Unlike SDS-PAGE, proteins are not deliberately given a uniform charge-to-mass ratio. Therefore migration depends on several properties.

These include:

Key difference: SDS-PAGE mainly separates proteins according to molecular size, whereas Native PAGE separates proteins according to a combination of charge, size and shape while attempting to preserve native structure.

5.9 Isoelectric Focusing — IEF

Isoelectric point (pI): The pI of a protein is the pH at which the protein has no net electrical charge.

Isoelectric focusing is an electrophoretic technique used to separate proteins according to their isoelectric points.

Isoelectric focusing
Figure 32. Isoelectric focusing showing proteins moving through a pH gradient and focusing at their isoelectric points.

Principle

In IEF, a stable pH gradient is established within the separation medium. A protein migrates through the gradient according to its net charge. As the protein approaches the pH corresponding to its pI, its net charge approaches zero. At this point its electrophoretic movement becomes very small and the protein focuses into a narrow band.

Importance of IEF

5.10 Two-Dimensional Gel Electrophoresis

Two-dimensional electrophoresis, commonly called 2D electrophoresis, combines two different separation principles. It is especially useful for resolving complex mixtures containing many proteins.

Two dimensional electrophoresis
Figure 33. Two-dimensional electrophoresis: first dimension by isoelectric focusing followed by second-dimensional SDS-PAGE.

First Dimension

Proteins are separated by isoelectric focusing according to their isoelectric points.

Second Dimension

The focused proteins are transferred to an SDS-polyacrylamide gel and separated mainly according to molecular mass.

Protein mixture → Isoelectric focusing → Separation by pI → SDS-PAGE → Separation by molecular mass → Two-dimensional protein pattern

Applications

Advantages

Limitations

5.11 General Electrophoresis Setup

Electrophoresis apparatus
Figure 34. General electrophoresis apparatus showing power supply, electrodes, buffer chamber and gel.

5.12 Comparison of Major Electrophoretic Techniques

Technique Major molecule Main basis of separation Important use
Agarose electrophoresis DNA/RNA Mainly size DNA fragment analysis
SDS-PAGE Proteins Mainly molecular mass Protein size and purity
Native PAGE Proteins Charge, size and shape Native protein analysis
IEF Proteins Isoelectric point Protein pI analysis
2D electrophoresis Proteins pI followed by molecular mass Proteomics

Important Examination Questions — Unit 5

2 marks: Define electrophoresis.
2 marks: What is electrophoretic mobility?
2 marks: Define isoelectric point.
5 marks: Explain the principle and applications of agarose gel electrophoresis.
5 marks: Differentiate SDS-PAGE and Native PAGE.
5 marks: Explain isoelectric focusing.
10 marks: Describe electrophoresis, its principle, instrumentation, types and applications.
10 marks: Explain SDS-PAGE in detail with principle, procedure, applications, advantages and limitations.
10 marks: Describe two-dimensional gel electrophoresis and explain its importance in proteomics.

UNIT 6 — CHROMATOGRAPHIC TECHNIQUES

6.1 Introduction to Chromatography

Definition: Chromatography is a separation technique in which components of a mixture are distributed between a stationary phase and a mobile phase, resulting in differential migration or retention of the components.

Chromatography is one of the most versatile techniques used in biological research. It can be used to separate, identify, purify and sometimes quantify components of complex biological mixtures.

Proteins, peptides, amino acids, nucleotides, carbohydrates, lipids, pigments, drugs, metabolites and many other compounds can be analyzed using appropriate chromatographic methods.

6.2 Basic Principle

Chromatographic separation occurs because different molecules interact differently with the stationary phase and mobile phase. A molecule that has a stronger interaction with the stationary phase generally moves more slowly, while a molecule that remains more strongly associated with the mobile phase moves more rapidly.

Basic principle of chromatography
Figure 35. Basic chromatographic principle showing differential interaction with stationary and mobile phases.

6.3 Important Components

6.4 Classification of Chromatography

6.5 Paper Chromatography

Paper chromatography is a simple chromatographic method historically used for separating small molecules such as amino acids, sugars and pigments.

Paper chromatography
Figure 36. Paper chromatography showing sample spot and separation of components along the paper.

Principle

The sample components distribute differently between the stationary environment associated with the paper and the moving solvent. Different components therefore migrate different distances.

Rf Value

The relative mobility of a component can be expressed using the retardation factor, commonly written as Rf.

Rf = Distance travelled by solute / Distance travelled by solvent front

Important Points About Rf

Applications

6.6 Thin-Layer Chromatography — TLC

Thin-layer chromatography is a simple and rapid technique in which the stationary phase is coated as a thin layer over a solid support. Silica gel and alumina are commonly used stationary phases.

Thin layer chromatography
Figure 37. Thin-layer chromatography showing sample application, development chamber and separated spots.

General Procedure

  1. Prepare or obtain the TLC plate.
  2. Draw a light baseline near the lower edge.
  3. Apply a small amount of sample.
  4. Place the plate in the developing chamber.
  5. Allow the solvent to move upward.
  6. Remove the plate before the solvent reaches the top.
  7. Mark the solvent front.
  8. Visualize separated compounds.
  9. Calculate Rf values where appropriate.

Applications

6.7 Column Chromatography

Column chromatography is a technique in which the stationary phase is packed into a column and the mobile phase passes through it. Components of a mixture move at different rates and are collected in separate fractions.

Column chromatography
Figure 38. Column chromatography showing packed stationary phase, sample layer, mobile phase and collected fractions.

General Steps

  1. Prepare and pack the column.
  2. Equilibrate the stationary phase.
  3. Apply the sample carefully.
  4. Add mobile phase.
  5. Allow components to migrate.
  6. Collect fractions.
  7. Analyze the fractions.
  8. Pool desired fractions when appropriate.

Applications

6.8 Ion-Exchange Chromatography

Ion-exchange chromatography separates molecules according to their electrical charge. The stationary phase contains charged functional groups that interact with oppositely charged molecules.

Ion exchange chromatography
Figure 39. Ion-exchange chromatography showing binding and elution of charged proteins.

Types

Elution

Bound molecules may be released by changing the salt concentration, changing pH or modifying other conditions that alter electrostatic interactions.

Applications

6.9 Size-Exclusion Chromatography

Size-exclusion chromatography separates molecules primarily according to their hydrodynamic size. It is also known as gel filtration when aqueous systems are used.

Gel filtration chromatography
Figure 40. Size-exclusion chromatography showing large and small molecules moving through porous beads.

Principle

The stationary phase contains porous beads. Large molecules cannot enter many of the pores and therefore travel through the column more directly. Smaller molecules can enter more pores and consequently take a longer effective path through the column.

Key concept: In size-exclusion chromatography, larger molecules generally elute before smaller molecules because large molecules are excluded from more of the internal pore volume.

Applications

6.10 Affinity Chromatography

Affinity chromatography is a highly selective purification technique based on a specific biological interaction between a target molecule and a ligand attached to the stationary phase.

Affinity chromatography
Figure 41. Affinity chromatography showing specific binding of the target molecule to an immobilized ligand.

Principle

The target molecule specifically recognizes and binds to an immobilized ligand. Other molecules that do not bind specifically pass through the column. The target is subsequently eluted by changing appropriate conditions.

Examples

Advantages

Limitations

6.11 High-Performance Liquid Chromatography — HPLC

High-performance liquid chromatography is an advanced chromatographic technique in which a liquid mobile phase is forced through a packed stationary phase under relatively high pressure.

High performance liquid chromatography
Figure 42. Basic HPLC system showing solvent reservoirs, pump, injector, column and detector.

Main Components of HPLC

  1. Solvent reservoirs
  2. Degassing system
  3. High-pressure pump
  4. Sample injector
  5. Chromatographic column
  6. Detector
  7. Data acquisition system

Working

The mobile phase is delivered under pressure through the chromatographic column. The sample is introduced into the mobile phase and passes through the stationary phase. Components interact differently with the column and therefore elute at different times.

Applications of HPLC

6.12 Gas Chromatography — GC

Gas chromatography is a chromatographic technique used primarily for compounds that can be vaporized without decomposition or that can be converted into suitable volatile derivatives.

Gas chromatography system
Figure 43. Basic gas chromatography system showing carrier gas, injector, column, oven and detector.

Major Components

Applications

6.13 General Chromatographic Workflow

Chromatography workflow
Figure 44. General workflow of chromatographic separation.
Sample preparation → Selection of chromatographic method → Equilibration → Sample application → Separation → Detection → Fraction collection → Analysis

6.14 Comparison of Important Chromatographic Techniques

Technique Major basis Common application
Paper chromatography Differential partition/interaction Amino acids, pigments
TLC Adsorption/partition Small molecules and reaction monitoring
Column chromatography Differential interaction with stationary phase Preparative purification
Ion exchange Charge Protein purification
Size exclusion Hydrodynamic size Protein separation and desalting
Affinity Specific biological interaction Highly selective protein purification
HPLC Different retention in high-pressure liquid system Analytical and preparative applications
GC Volatility and interaction with stationary phase Volatile compounds

Important Examination Questions — Unit 6

2 marks: Define chromatography.
2 marks: What is stationary phase?
2 marks: Define Rf value.
5 marks: Explain the principle and procedure of TLC.
5 marks: Explain ion-exchange chromatography.
5 marks: Describe size-exclusion chromatography.
10 marks: Describe chromatography and classify the major chromatographic techniques with their principles and applications.
10 marks: Explain affinity chromatography in detail with principle, procedure, applications, advantages and limitations.
10 marks: Describe HPLC with principle, instrumentation, working and applications.
10 marks: Explain different chromatographic methods used in biological research.

IMPORTANT CALCULATIONS AND RESULT INTERPRETATION

1. Calculation of Rf Value

A common numerical question in chromatography involves calculation of the Rf value.

Rf = Distance travelled by compound / Distance travelled by solvent front

Example 1

Suppose a compound travels 4.0 cm from the origin while the solvent front travels 8.0 cm.

Rf = 4.0 / 8.0 = 0.50

Therefore, the Rf value of the compound is 0.50.

Example 2

A compound travels 3.5 cm while the solvent front travels 7.0 cm.

Rf = 3.5 / 7.0 = 0.50

Therefore, the Rf value is 0.50.

Example 3

An unknown sample has travelled 5.2 cm and the solvent front has travelled 8.0 cm.

Rf = 5.2 / 8.0 = 0.65

The Rf value is therefore 0.65.

Calculation Tips for Rf Numericals

  • Always measure distance from the original spotting line.
  • Measure the solvent front separately.
  • Do not use the total length of the TLC plate.
  • Rf has no physical unit.
  • Under identical experimental conditions, Rf values can help compare unknown and standard compounds.
  • Rf should normally be between 0 and 1.

2. Interpretation of Electrophoresis Bands

When interpreting an electrophoresis gel, the position, intensity and number of bands should be considered.

3. Approximate Protein Molecular Mass from SDS-PAGE

In SDS-PAGE, the migration of a protein can be compared with molecular mass standards. A calibration plot can be prepared using the migration distance or relative mobility of known standards.

A common approach is to plot the logarithm of molecular mass against relative migration and use the resulting calibration relationship to estimate the molecular mass of an unknown protein.

Examination point: A molecular-weight marker should be run alongside unknown protein samples when estimating molecular mass by SDS-PAGE.

4. Chromatographic Elution Profile

In instrumental chromatography, separated compounds may appear as peaks on a chromatogram. Each peak represents a detected component or group of components under the experimental conditions.

Important features include:

5. Retention Time

Retention time: The time between sample injection and the detection of a particular chromatographic component at the detector.

Retention time can be useful for identifying compounds by comparison with standards analyzed under the same conditions. Identification should not normally be based on retention time alone when definitive identification is required.

MASTER COMPARISON — ELECTROPHORESIS VS CHROMATOGRAPHY

Feature Electrophoresis Chromatography
Basic principle Migration in an electric field Differential distribution between stationary and mobile phases
Major driving factor Electrical field Movement of mobile phase
Common molecules DNA, RNA, proteins Proteins, metabolites, drugs, pigments and many other compounds
Common supporting system Agarose or polyacrylamide gel Column, plate, paper or specialized chromatographic column
Separation basis Charge, size, shape or pI depending on method Charge, size, affinity, adsorption, partition or volatility
Major application Analysis of nucleic acids and proteins Separation, purification and analysis of many biological compounds

SECTION 2 — QUICK REVISION POINTS

Electrophoresis

  • Electrophoresis separates charged molecules using an electric field.
  • DNA is negatively charged because of its phosphate backbone.
  • Agarose is commonly used for DNA and RNA separation.
  • PAGE provides high-resolution separation.
  • SDS-PAGE mainly separates proteins according to molecular mass.
  • Native PAGE preserves more of the native properties of proteins.
  • IEF separates proteins according to isoelectric point.
  • 2D electrophoresis combines IEF and SDS-PAGE.

Chromatography

  • Chromatography uses stationary and mobile phases.
  • Different molecules interact differently with the two phases.
  • Rf is important in planar chromatography.
  • Ion-exchange chromatography separates according to charge.
  • Size-exclusion chromatography separates according to hydrodynamic size.
  • Affinity chromatography is based on specific biological interactions.
  • HPLC uses high-pressure liquid flow through a chromatographic column.
  • GC is particularly useful for volatile compounds.

High-Yield Long-Answer Topics

  1. Principle, instrumentation and applications of electrophoresis.
  2. Agarose gel electrophoresis.
  3. SDS-PAGE.
  4. Native PAGE and its comparison with SDS-PAGE.
  5. Isoelectric focusing.
  6. Two-dimensional electrophoresis.
  7. General principles of chromatography.
  8. Ion-exchange chromatography.
  9. Size-exclusion chromatography.
  10. Affinity chromatography.
  11. HPLC.
  12. Comparison of chromatographic techniques.

SECTION 2 — EXAM-ORIENTED QUESTION BANK

Very Short Questions — 2 Marks

  1. Define electrophoresis.
  2. What is electrophoretic mobility?
  3. What is agarose?
  4. Define SDS-PAGE.
  5. What is Native PAGE?
  6. Define isoelectric point.
  7. What is isoelectric focusing?
  8. What is two-dimensional electrophoresis?
  9. Define chromatography.
  10. What is stationary phase?
  11. What is mobile phase?
  12. Define Rf value.
  13. What is ion-exchange chromatography?
  14. What is gel filtration?
  15. Define affinity chromatography.
  16. What is HPLC?
  17. What is gas chromatography?
  18. Define retention time.

Short Questions — 5 Marks

  1. Explain the principle of agarose gel electrophoresis.
  2. Describe the basic components of an electrophoresis apparatus.
  3. Explain SDS-PAGE.
  4. Differentiate Native PAGE and SDS-PAGE.
  5. Explain isoelectric focusing.
  6. Describe two-dimensional electrophoresis.
  7. Explain paper chromatography.
  8. Describe thin-layer chromatography.
  9. Explain ion-exchange chromatography.
  10. Describe size-exclusion chromatography.
  11. Explain affinity chromatography.
  12. Describe the major components of HPLC.
  13. Explain gas chromatography.

Long Questions — 10 Marks

  1. Describe electrophoresis in detail, including its principle, instrumentation, factors affecting migration, major types and applications.
  2. Explain agarose gel electrophoresis with principle, gel preparation, sample loading, working, applications, advantages and limitations.
  3. Describe SDS-PAGE in detail and explain how proteins are separated according to molecular mass.
  4. Explain Native PAGE and compare it with SDS-PAGE.
  5. Describe isoelectric focusing and explain its principle and applications.
  6. Explain two-dimensional gel electrophoresis with a suitable diagram and discuss its importance in proteomics.
  7. Define chromatography and describe its general principle, components and classification.
  8. Explain ion-exchange chromatography in detail with principle, types, working and applications.
  9. Describe size-exclusion chromatography with principle, working and applications.
  10. Explain affinity chromatography in detail with principle, working, applications, advantages and limitations.
  11. Describe HPLC with principle, instrumentation, working and applications.
  12. Discuss important chromatographic techniques used in biological research.
How to write a 10-mark answer: For a major Bioinstrumentation question, do not stop after writing only the definition and principle. A strong university answer should normally include: Definition → Principle → Diagram → Components/Instrumentation → Working/Procedure → Factors affecting the technique → Applications → Advantages → Limitations → Conclusion. For comparison questions, use a table after explaining both techniques.
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