Adsorption chromatography column showing stationary and mobile phases

Adsorption Chromatography – Principle, Types, Procedure, Applications & Advantages

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Introduction

  • Adsorption chromatography is a type of liquid-solid or gas-solid chromatography where compounds are separated based on how strongly they bind (adsorb) to a solid surface.
  • The stationary phase is a solid adsorbent, and the mobile phase can be a liquid or a gas.
  • Separation happens because different molecules interact with the stationary phase with varying strengths, leading to different movement speeds through the column or surface.
  • This technique was first discovered in 1903 by Mikhail Tswett, a Russian botanist, during his research on plant pigments.
  • Today, adsorption chromatography remains one of the oldest yet widely used chromatographic techniques in research, pharmaceuticals, environmental testing, and industry.

Principle of Adsorption Chromatography

  • Works on the differential adsorption principle:
    • The stationary phase is a solid adsorbent.
    • The mobile phase is either a liquid or a gas.
    • Molecules in the sample compete with the mobile phase for binding sites on the stationary phase.
  • Forces involved in adsorption:
    • Van der Waals forces
    • Hydrogen bonding
    • Dipole–dipole interactions
    • Electrostatic interactions
  • Polarity matters:
    • Polar molecules stick more strongly to polar stationary phases.
    • Non-polar molecules pass through faster.
  • Key concept:
    • Stronger binding affinity → slower movement → later elution.
    • Weaker binding affinity → faster movement → earlier elution.

Phases in Adsorption Chromatography

1. Stationary Phase

  • Solid adsorbent that binds molecules through weak physical forces.
  • Common adsorbents:
    • Polar acidic: Silica gel – best for separating polar basic compounds.
    • Polar basic: Alumina, Florisil – best for polar acidic compounds.
    • Non-polar: Charcoal, polystyrene – best for non-polar compounds.

2. Mobile Phase

  • Moves through the stationary phase and carries sample molecules.
  • Can be:
    • Liquid → Solid-liquid adsorption chromatography
    • Gas → Gas-solid adsorption chromatography
  • Selection criteria:
    • Polarity
    • Solvent strength
    • Compatibility with stationary phase and sample

Types of Adsorption Chromatography

A. Based on Stationary Phase Polarity

  1. Normal Phase Adsorption Chromatography
    • Stationary phase: Polar
    • Mobile phase: Non-polar
    • Polar compounds stick longer; non-polar compounds elute faster.
    • Often uses a gradient elution where solvent polarity increases over time.
  2. Reversed Phase Adsorption Chromatography
    • Stationary phase: Non-polar
    • Mobile phase: Polar
    • Non-polar compounds stick longer; polar compounds elute faster.
    • Solvent polarity decreases gradually in gradient elution.

B. Based on Mobile Phase

  1. Solid-Liquid Adsorption Chromatography
    • Mobile phase: Liquid
    • Used for separating non-volatile compounds.
  2. Solid-Gas Adsorption Chromatography
    • Mobile phase: Gas (e.g., nitrogen, helium)
    • Used for separating volatile substances.

Forms of Adsorption Chromatography

1. Thin Layer Chromatography (TLC)

  • Adsorbent layer (e.g., silica gel) coated on a glass/plastic plate.
  • Sample spotted near one end; solvent moves up by capillary action.
  • Components separate based on affinity for stationary vs. mobile phase.
  • Used for quick qualitative analysis.

2. Paper Chromatography

  • Stationary phase: Paper (cellulose fibers).
  • Mobile phase: Liquid solvent.
  • Common for separating pigments, amino acids, and small molecules.

3. Column Chromatography

  • Stationary phase packed in a vertical column.
  • Mobile phase flows downward, carrying the sample through.
  • Compounds elute at different times depending on binding strength.

4. Gas-Solid Chromatography

  • Stationary phase: Solid adsorbent like alumina or silica.
  • Mobile phase: Inert gas.
  • Used for separating gases or volatile liquids.

Procedure – Steps in Adsorption Chromatography

A. Column Method

  1. Column Preparation
    • Clean and dry the glass column.
    • Pack with stationary phase, avoiding air bubbles.
  2. Mobile Phase Selection
    • Choose based on sample polarity and separation goals.
  3. Sample Loading
    • Apply sample gently to prevent disturbance.
  4. Elution
    • Pass mobile phase continuously through column.
  5. Fraction Collection
    • Collect eluted components in test tubes for analysis.

B. Thin Layer Method

  1. Prepare TLC chamber and saturate with solvent vapor.
  2. Coat glass plate with thin adsorbent layer.
  3. Spot sample on baseline.
  4. Place plate in chamber (baseline above solvent).
  5. Allow solvent to rise; remove plate and dry.
  6. Visualize spots under UV light or iodine vapor.
  7. Calculate Rf value = (Distance moved by sample) ÷ (Distance moved by solvent).

Factors Affecting Adsorption Chromatography

  • Nature of adsorbent: Smaller particles, higher surface area = better separation.
  • Solvent properties: Polarity, pH, ionic strength.
  • Column dimensions: Length and diameter influence resolution.
  • Temperature: Can change adsorption strength.
  • Flow rate: Too fast reduces resolution; too slow increases time.

Applications of Adsorption Chromatography

  • Organic compound separation
  • Non-ionic, water-insoluble compounds (e.g., triglycerides, vitamins, drugs)
  • Amino acid, carbohydrate, and protein purification
  • Hormone and antibiotic detection in biological fluids
  • Food & pharmaceutical quality control
  • Forensic analysis (drug detection, ink analysis)
  • Phytochemical separation from plant extracts
  • Lipid and steroid separation
  • Enantiomer separation
  • Environmental analysis (pesticides, herbicides)

Advantages of Adsorption Chromatography

  • Works with a wide variety of mobile phases.
  • Effective for mixtures that other methods cannot separate.
  • High versatility.
  • Can be adapted to analytical or preparative scales.

Limitations of Adsorption Chromatography

  • Can be time-consuming.
  • Requires more skill and precision.
  • Low reproducibility compared to some modern techniques.
  • Equipment and adsorbents can be costly.

Troubleshooting & Safety

  • Ensure proper packing of stationary phase.
  • Choose correct solvent polarity.
  • Wear gloves and safety glasses.
  • Handle flammable solvents with care.
  • Dispose of chemicals properly.

Recent Advances

  • Development of high-purity adsorbents for better resolution.
  • Automation and miniaturization for faster analysis.
  • Integration with spectrophotometry for instant detection.
  • Use of eco-friendly adsorbents and solvents.

Adsorption chromatography is a versatile, reliable, and widely used separation technique.
From pharmaceuticals to environmental science, it plays a vital role in research, quality control, and industrial processes.
With ongoing innovations, it continues to evolve into a faster, greener, and more efficient method for compound separation.

Frequently Asked Questions (FAQ)

Q1. What is adsorption chromatography used for?
It is used to separate and purify compounds such as pigments, lipids, amino acids, and drugs based on their adsorption to a solid stationary phase.

Q2. What is the principle of adsorption chromatography?
It works on the principle that molecules bind to a solid adsorbent with different strengths, causing them to move at different speeds and separate from each other.

Q3. What are the main types of adsorption chromatography?
The main types include normal phase, reversed phase, solid-liquid adsorption chromatography, and solid-gas adsorption chromatography.

Q4. What is the difference between normal phase and reversed phase adsorption chromatography?
Normal phase uses a polar stationary phase and non-polar mobile phase, while reversed phase uses a non-polar stationary phase and polar mobile phase.

Q5. Is TLC an example of adsorption chromatography?
Yes, thin layer chromatography (TLC) is a form of adsorption chromatography where the stationary phase is coated on a flat plate.

Q6. What are the limitations of adsorption chromatography?
It can be time-consuming, sometimes has low reproducibility, and requires skilled operation to avoid errors.

Q7. Who discovered adsorption chromatography?
It was first demonstrated by Mikhail Tswett in 1903 during his studies on plant pigments.

References 

  1. Manandhar, S. and Sharma, S. (2006). Practical Approach to Microbiology. 1st Edition. National Book Centre. 
  2. Banjara, M.R. and Thapa Shrestha, U. (2021). Instrumentation in Microbiology. Garuda Publications.
  3. Wilson, K. and Walker, J. (Ed.). (2010). Principles and Techniques of Biochemistry and Molecular Biology. Seventh Edition. Cambridge University Press. 
  4. Sharma, K. (2024, March 8). Adsorption Chromatography: Definition, principle, procedure, types, applications. Science Info. https://scienceinfo.com/adsorption-chromatography-principle-procedure-types-applications/
  6. Adsorption Chromatography – Principle, Procedure, experiment, Application, Types of Adsorption chromatography. BYJUS. https://byjus.com/chemistry/adsorption-chromatography/
  7. A short note on adsorption chromatography. (2022, April 26). Unacademy. https://unacademy.com/content/neet-ug/study-material/chemistry/a-short-note-on-adsorption-chromatography/
  8. Adsorption chromatography. VEDANTU. https://www.vedantu.com/chemistry/adsorption-chromatography

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