Study Notes On The Principles And Application Of Paper Chromatography

PRINCIPLES AND APPLICATION OF PAPER CHROMATOGRAPHY

INTRODUCTION TO CHROMATOGRAPHY

• The term chromatography derives from Greek:

  • "Chroma" meaning color

  • "Graphein" meaning to write

• Chromatography is an essential analytical technique that separates, identifies, and quantifies components of a mixture.

• Principle: Solutes are separated through a dynamic differential migration process based on their varying affinities for a stationary phase (solid or liquid) and a mobile phase (liquid or gas). This differential partitioning leads to effective separation of complex mixtures, allowing for purification, compositional analysis, or identification of unknown substances.

TYPES OF CHROMATOGRAPHY

1. Paper Chromatography

2. Thin Layer Chromatography (TLC)

3. Column Chromatography

4. Gas Chromatography (GC)

5. Liquid Chromatography (LC)

6. High-Performance Liquid Chromatography (HPLC)

PAPER CHROMATOGRAPHY

• Initiated by German scientist Christian Friedrich Schonbein in 1865.

• Discovered in its current form by Sehon Ben in 1961, and further developed by Synge and Martin in 1943.

• Paper chromatography (PC) involves analyzing an unknown substance through the flow of solvents on specially designed filter paper.

• It is a form of planar chromatography using filter paper made primarily of cellulose as the stationary phase. Cellulose is a polar material due to its abundant hydroxyl groups, which allows it to readily absorb water, forming a quasi-stationary liquid phase.

• PC is recognized as the simplest and least expensive type of chromatography.

• It requires very small quantities of the sample for analysis.

PRINCIPLE

• The basic separation principle is predominantly partitioning, where components of a mixture distribute themselves between two immiscible phases.

• Partitioning Process: Solute molecules interact with the stationary phase and distribute themselves dynamically between the stationary and mobile phases. The extent of interaction (affinity) with each phase dictates how fast a solute moves, leading to separation.

• PC encompasses:
  
  i. Partition chromatography
  
  ii. Adsorption chromatography

Partition Chromatography

• Separation of components occurs between two liquid phases:

  • One liquid phase is water/moisture, tightly retained in the cellulose pores of the filter paper, acting as the stationary phase due to the polar nature of cellulose.

  • The second phase (mobile phase) consists of organic solvents or buffers.

  • Differences in affinities towards the water (stationary phase) and mobile phase solvents result in separation through capillary action in the filter paper.

Adsorption Chromatography

• In this method, separation occurs between solid and liquid phases:

  • The solid surface of the paper (impregnated with silica or alumina) serves as the stationary phase.

  • The liquid solvent acts as the mobile phase.

• Although the main working principle of paper chromatography is partitioning, adsorption is widely applied in pharmaceuticals.

INSTRUMENTATION OF PAPER CHROMATOGRAPHY

1. Stationary Phase & Papers Used:

• Whatman Filter Papers of various grades:

  • No.1, No.2, No.3, No.4, No.20, No.40, No.42.

  • Composition: 98-99% $\alpha$-cellulose, 0.3-1% $\beta$-cellulose.

• Other modified papers include:

  • Acid or base-washed filter paper.

  • Glass fiber type paper.

  • Hydrophilic and hydrophobic papers modified with substances like methanol or acetylation.

  • Impregnation with silica or ion exchange resins.

1. Mobile Phase:

• Pure solvents, buffer solutions, or mixtures of solvents can be employed. The choice of mobile phase is crucial and depends on the polarity of the analytes and the stationary phase, often requiring optimization through experimentation. Common organic solvents include ethanol, n-butanol, propanol, acetone, and benzene, often mixed with water or weak acids/bases to finely tune elution properties.

1. Chromatographic Chamber:

• Materials: Glass, plastic, or stainless steel (glass is preferred).

• Varying dimensions based on paper length and development type.

• Chamber atmosphere must be saturated with solvent vapor to prevent evaporative effects and ensure a uniform solvent front.

STEPS IN PAPER CHROMATOGRAPHY

1. Selection of Solid Support:

• Use fine quality cellulose paper with defined porosity for high resolution and reduced sample diffusion.

1. Selection of Mobile Phase:

• Different combinations of solvents may be used based on the analysis.

• Example: Butanol: Acetic acid: Water in a 12:3:5 ratio is effective for amino acids.

1. Saturation of Tank:

• The inner tank wall is wrapped with filter paper before introducing the solvent for optimal resolution.

1. Sample Preparation and Loading:

• Solid samples should be dissolved in a suitable solvent.

• Apply a small, concentrated spot (typically 2-20 µl of the solution) to the baseline using a micropipette. This concentration is critical to prevent band broadening and ensure clear separation, and the spot should be air-dried to reduce initial diffusion.

1. Development of the Chromatogram:

• Immerse the sample-loaded paper into the solvent, not exceeding 1 cm in height, and wait until the solvent front nears the paper's edge.

1. Drying of Chromatogram:

• Mark the solvent front and dry the chromatogram in a dry cabinet or oven.

1. Detection:

• Colorless analytes can be visualized using reagents like iodine vapor or ninhydrin.

• Fluorescent and radiolabeled analytes require measurement of fluorescence and radioactivity, respectively.

METHODOLOGY FOR PAPER CHROMATOGRAPHY

• Obtain a chromatography jar, fresh spinach, and chromatography paper.

• Prepare the paper by cutting the tip to form a triangle shape.

• Draw a starting line approximately 1 cm from the triangle tip.

• Use a capillary tube to place a drop of extract on the starting line.

• Pour approximately 1 cm of chromatography solvent into the jar.

• Hang the paper in the jar ensuring the triangle tip dips into the solvent.

• Allow solvent migration until it approaches within 1 cm from the top of the paper.

MODES/TYPES OF PAPER CHROMATOGRAPHY

1. Ascending Development:

• The solvent flows against gravity; spots remain at the bottom of the paper, placed in a chamber with mobile phase solvent at the base.

1. Descending Development:

• Conducted in a special chamber with the solvent holder at the top; the solvent flows downward over the paper.

1. Ascending – Descending:

• A hybrid technique where the solvent first ascends then descends, thus extending the length of separation.

1. Circular / Radial (Horizontal):

• The spot is placed at the center of a circular paper with solvent moving uniformly from a wick at the center.

1. Two-Dimensional Chromatography:

• Utilizes two perpendicular solvent developments to separate mixtures with similar Rf values.

Rf VALUES

• The Rf value (Retention factor) is a measure of the distance traveled by a component relative to the distance traveled by the solvent front.

• Formula:
  
  $Rf = \frac{\text{Distance traveled by solute/component}}{\text{Distance traveled by solvent}}$

• Rf values are dimensionless and always range between $0$ and $1$. An Rf value of $0$ indicates the solute remained at the origin, while $1$ means it traveled with the solvent front. Rf values remain consistent for specific compounds under controlled parameters such as paper quality and solvent composition.

ADVANTAGES OF PAPER CHROMATOGRAPHY

a. Simple and rapid procedures.

b. Requires minimal sample quantities.

c. Cost-effective compared to other chromatography methods.

d. Capable of identifying unknown inorganic and organic compounds.

e. Space-efficient compared to alternative analytical methods.

f. Possesses excellent resolution characteristics.

LIMITATIONS OF PAPER CHROMATOGRAPHY

a. Not suitable for large sample quantities.

b. Ineffective for quantitative analysis of complex mixtures.

c. Reduced accuracy compared to HPLC or HPTLC techniques.
d. APPLICATIONS OF PAPER CHROMATOGRAPHY

• Separation of mixtures containing polar and non-polar compounds.

• Separation of amino acids.

• Determining organic biochemical compounds in urine.

• Detection of drugs and doping agents