Introduction to Chromatographic Methods

• Chromatography separates the components of a mixture by exploiting the interactions of components with both the mobile phase and the stationary phase.
• A basic example of chromatography is paper chromatography.
• The class will include a short demonstration of chromatography techniques.

Thin Layer Chromatography (TLC)

Definition and Importance

• Thin layer chromatography (TLC) is a key technique for identification and separation of mixtures of organic compounds.
• Utilizes of TLC includes:
  • Identification of components within a mixture using appropriate standards.
  • Isolation of components from mixtures.
  • Checking the progress of chemical reactions.
  • Analyzing the purity of a compound.

Mechanism of TLC

• In TLC, the components of the mixture are partitioned between:
  • Stationary phase: Typically silica gel (SiO2).
  • Mobile phase: Solvent that flows through the stationary phase.
• As the mobile phase ascends the TLC plate by capillary action, the components dissolve in the mobile phase and travel up the TLC plate.

Movement of Components

• Each component in the mixture moves up the TLC plate at different rates.
• The rate of movement depends on intermolecular forces:
  • Between the component and the stationary phase.
  • Between the component and the mobile phase.
• Silica gel, being polar, interacts strongly with polar compounds, which results in their slow movement up the TLC plates.
• Non-polar compounds interact weakly with the stationary phase and thus move faster up the plate.

Component Interaction

• The mobile phase is relatively less polar, which allows it to interact better with non-polar components.
• Therefore, non-polar analytes will move higher up the TLC plate as their interaction with the stationary phase is less strong compared to the mobile phase.

Experiment Demonstration

TLC Plate Preparation

• Cut a 7 × 10 cm TLC plate.
• Using a pencil, lightly draw a line 1.5 cm from the base of the plate.
• Draw another line 2 cm from the top of the plate.
• On the lower line, make dots at intervals of 1 cm apart.
• Apply spots of the samples or mixtures on each dot, labeling them accordingly.
• Fill a development chamber with the mobile phase to a depth of 0.5-1 cm.
• Cover the chamber and allow the mobile phase to rise to the top line before removing the plate.

Retention Factor (Rf)

Definition

• The retention factor (Rf) is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin).

Rf Values in Substance Identification

• Rf values can assist in the identification of substances through comparison with known compounds or standards.
• The Rf value is not constant and should only be compared among spots on the same plate that were run simultaneously.
• Compounds with the same Rf value may be identical; those with different Rf values are confirmed to be different compounds.

Interaction of Solutes with Stationary Phase

• The strength of interaction of compounds with the stationary phase increases with the increasing polarity of their functional groups:
  • Functional groups ranked from weak to strong interaction include:
  • -CH=CH₂ (non-polar)
  • -X
  • -OR
  • -CHO
  • -CO₂R
  • -NR₂
  • -NH₂
  • -OH
  • -CONR₂
  • -CO₂H (more polar).

Elution Strength of Mobile Phase

Definition

• The elution strength of the mobile phase (e) corresponds to the polarity of the solvent.
• It depends on the intermolecular forces between:
  • Mobile phase and analytes
  • Interaction between mobile and stationary phases.
  • More polar solvents possess stronger eluting capacities.

Measurement of Elution Strength

• The eluent strength (ε°), a measure of solute adsorption energy, has a value defined for pentane as 0 on bare silica.
• More polar solvents show higher eluent strength, causing solutes to elute more rapidly.

Elution Strength Data Table

• A table comparing different solvents regarding their molecular formula (MF), molecular weight (MW), boiling point (Bp in °C), density (g/mL), hazards, and elution strength properties:
  • Hexane:
  • MF: C6H14, MW: 86.17, Bp: 68.7, Density: 0.659 g/mL, Elution Strength (ε): 0.01, Hazard: Flammable, Toxic.
  • Toluene:
  • MF: C7H8, MW: 92.13, Bp: 110.6, Density: 0.867 g/mL, Elution Strength (ε): 0.22, Hazard: Flammable, Toxic.
  • Diethyl ether:
  • MF: C4H10O, MW: 74.12, Bp: 34.6, Density: 0.713 g/mL, Elution Strength (ε): 0.29, Hazard: Flammable, CNS Depressant.
  • Dichloromethane:
  • MF: CH2Cl2, MW: 84.94, Bp: 39.8, Density: 1.326 g/mL, Elution Strength (ε): 0.32, Hazard: Toxic, Irritant, Cancer suspect.
  • Ethyl Acetate:
  • MF: C4H8O2, MW: 88.10, Bp: 77.1, Density: 0.901 g/mL, Elution Strength (ε): 0.45, Hazard: Flammable, Irritant.
  • Acetone:
  • MF: C3H6O, MW: 58.08, Bp: 56.3, Density: 0.790 g/mL, Elution Strength (ε): 0.43, Hazard: Flammable, Irritant.
  • Butanone:
  • MF: C4H8O, MW: 72.10, Bp: 80.1, Density: 0.805 g/mL, Elution Strength (ε): 0.39, Hazard: Flammable, Irritant.
  • 1-Butanol:
  • MF: C4H10O, Bp: 117.7, Density: 0.810, Hazard: Flammable, Irritant.
  • Propanol:
  • MF: C3H8O, MW: 60.09, Bp: 82.3, Density: 0.785 g/mL, Elution Strength (ε): 0.63, Hazard: Flammable, Irritant.
  • Ethanol:
  • MF: C2H6O, Bp: 78.5, Density: 0.789 g/mL, Elution Strength (ε): 0.68, Hazard: Flammable, Irritant.
  • Methanol:
  • MF: CH3OH, MW: 32.04, Bp: 64.7, Density: 0.791 g/mL, Elution Strength (ε): 0.73, Hazard: Flammable, Toxic.
  • Water:
  • MF: H2O, MW: 18.02, Bp: 100.0, Density: 1.000 g/mL, Elution Strength (ε): >1, Hazard: non-toxic.

Elution Strength Calculation

• The elution strength of a mixture is assumed to be the weighted average of the elution strengths of its components:
  • $e_{onet} = e_{oA} imes ext{(mole ratio A)} + e_{oB} imes ext{(mole ratio B)}$
  • Where mole ratio A is defined as: $\frac{ ext{moles A}}{ ext{moles A + moles B}}$

Class Exercise

Elution Strength Calculation Example

• A TLC plate was developed in a diethyl ether/hexane (2:3) mixture. Total volume of mobile phase used was 10 mL.

  • Calculate the elution strength of the mixture:
  • For diethyl ether:
    • Density = 0.713 g/mL,
    • Volume in mixture = 4 mL,
    • Mass = $4 imes 0.713 = 2.852 ext{ g}$.
    • Moles of diethyl ether = $\frac{2.852}{74.12} = 0.0384$ moles.
  • For hexane:
    • Mass of hexane = Density × Volume = $0.659 ext{ g/mL} imes 6 ext{ mL} = 3.954 ext{ g}$.
    • Moles of hexane = $\frac{3.954 ext{ g}}{86.17 ext{ g/mol}} = 0.04589$ moles.
    • Total moles (diethyl ether + hexane) = $0.08429 ext{ moles}$.

• The final calculation for the elution strength of the mixture:

  • $e_{onet} = 0.29 imes \frac{0.0384}{0.08429} + 0.01 imes \frac{0.04589}{0.08429} = 0.1375$

Resolution in Chromatography

• Resolution refers to the separation of two analytes within a chromatogram.
• Good resolution is characterized by no overlap of analyte bands on the chromatogram.
• Overlapping bands may prevent accurate identification of substances.
• To improve resolution, one can:
  • Decrease the diameter of the analyte spot, which leads to decreased concentration of the analytes in that area.

Column Chromatography

• Column chromatography is a three-dimensional version of TLC.
• Same scientific principles apply as those in TLC:
  • Stationary Phase: Powdered silica gel.
  • Mobile Phase: Solvents similar to those used in TLC.
• Column chromatography is ideal for separation and collection of components from mixtures.

Recommended Reading Assignment

• Students are encouraged to read on the following topics:
  • Representation of a Gas Chromatography (GC) instrument.
  • Different components of GC instruments and their respective functions.
  • Types and functioning of detectors used in Gas Chromatography (GC).