Organic Chemistry Lab: Chromatography and Separation Techniques

Overview

• First-time liquid-liquid extraction experience in organic chemistry lab resulting in extremely high and unrealistic percent yield.
• Focus on understanding what went wrong in the process and how chromatography works.
• How these topics are tested on the MCAT.

Key Topics Covered

• Chromatography and Separation Techniques (high-yield for MCAT).
• High-yield topics: Nucleic Acids, Amino Acids, Protein Structure, Intermolecular Forces.

Types of Chromatography

• Liquid-Liquid Extraction: Separating organic and inorganic layers based on solubility.
• Distillation: Separation based on different boiling points.
• Column Chromatography: Includes ion exchange, affinity, size exclusion.
• Gas Chromatography (GC): Separation based on boiling points/volatility.
• Thin Layer Chromatography (TLC): Uses a silica sheet, separates based on polarity; involves mobile and stationary phases.
• High-Pressure Liquid Chromatography (HPLC): Similar rules to TLC but in high pressure. Often mentioned in third-party exams.
• Ion Exchange Chromatography: Separates based on charge using charged liquid and charged polymers (beads).

Important Details for Each Technique

Thin Layer Chromatography (TLC)

• Components:
  • Mobile Phase: Typically nonpolar (e.g., hexane).
  • Stationary Phase: Silica gel sheet (polar).
• Retention Factor (RF): Measurement used in TLC (distance solute traveled divided by distance solvent front traveled).
• Solvent Front: The farthest point reached by the solvent.
• Application: Identifying the number of molecules present in a solution.
• Reverse Phase TLC: Involves polar mobile phase and nonpolar stationary phase.

Gas Chromatography (GC)

• Components: Separates based on boiling point.
• Used for: Volatile compounds.

Ion Exchange Chromatography

• Components: Uses charged beads (polymers) in the column.
• Separation Principle: Based on the charge of the molecules in the mobile phase.
• Types:
  • Cation Exchange Chromatography: Selects for positively charged ions.
  • Anion Exchange Chromatography: Selects for negatively charged ions.
• Process:
  • Solutions pass through beads and separate based on charge attraction.
  • Positive molecules elute first in an anion exchange because they don’t stick to the positively charged beads.
  • Negative molecules elute last after being washed out by a salt solution.
• Term 'Eluent': The liquid that exits the column during the chromatography process.

Size Exclusion Chromatography

• Components: Porous beads in the column.
• Separation Principle: Based on molecule size.
• Process:
  • Large molecules elute first as they navigate around the beads.
  • Medium-sized molecules elute second, passing through some pores.
  • Small molecules elute last, passing through many pores.
• Also Known As: Gel filtration chromatography.

Example Questions (MCAT-style)

Question 1

• Setup: Separation of compounds using reverse-phase HPLC.
• Concept Tested: Understanding of polarity impacts on elution time.
• Answer Example: Compound with higher polarity sticks to the polar mobile phase and elutes faster.

Question 2

• Setup: Relationship between steroid and peptide hormones and separation in hexane-water mix.
• Concept Tested: Understanding of polarity and solubility in extraction methods.
• Answer Example: Steroid hormone (nonpolar) in hexane phase; Peptide hormone (polar) in aqueous phase.

Question 3

• Setup: Protein charge and affinity for ion exchange column.
• Concept Tested: Understanding of protein interaction with ion exchange columns.
• Answer Example: Proteins with the largest negative net charge bind strongly to an anion exchange column.

Tips for MCAT Preparation

• Focus on main separation principles: polarity, charge, size, boiling point.
• Know key terms for each chromatography technique (e.g., RF value for TLC, eluent in column chromatography).
• Use simplified examples and practical lab experiences to retain concepts (e.g., lab stories, visualizing the setup).