Chromatography

FOOD ANALYSIS: Principles of Chromatography

Instructor Information

• Name: B. Pam Ismail

• Email: bismailm@umn.edu

• Course: FSCN 146

Introduction to Food Analysis and Chromatography

Key Topic: Column interaction

Chromatography is a vital separation technique used in food analysis to separate mixtures into their components based on partitioning or distribution between stationary and mobile phases.

Intermolecular Interactions in Adsorption Chromatography

Key Forces Responsible for Chromatography

• Electrostatic Forces: Interaction between charged surfaces and ions.

• Hydrogen Bonds: Attractive interactions between a hydrogen atom covalently bonded to an electronegative atom and another electronegative atom.

• Hydrophobic Interactions: Tendencies of non-polar substances to aggregate in aqueous solutions, minimizing their exposure to polar solvents.

Reverse-Phase Chromatography

Characteristics

• Polar compounds elute first.

• Non-polar compounds elute first.

• The mobile phase is often a polar liquid.

Historical Perspective of Chromatography

Key Figures

• David Day: An American geologist who contributed to the understanding of crude petroleum in relation to fuller earth.

• Mikhail Tsvet: A Russian botanist credited with the discovery of chromatography, by using it to separate leaf pigments through columns packed with chalk.

Development Timeline

• 1940s: Evolution of partition chromatography and paper chromatography.

• 1960s: Gas chromatography developed largely due to breakthroughs in the petroleum industry, followed by advances in liquid chromatography and supercritical fluid chromatography (SFC).

Applications of Chromatography

Primary Uses

• Actual means of analysis of components in food.

• Preliminary sample clean-up.

• Concentration of components of interest.

Definitions and Key Concepts

Key Questions and Answers

1. What is chromatography?

   • A separation technique used in food analysis based on the partition or distribution of solutes.

2. Stationary Phase vs. Mobile Phase

   • The stationary phase is the phase that does not move, usually a solid or a liquid coated onto a solid. The mobile phase is the solvent that carries the solute through the stationary phase, aiding in separation.

3. Extraction

   • The transfer of a solute from one liquid phase to another, used for sample cleanup and concentration.

4. Types of Chromatography

   • Includes various methods such as gas chromatography (GC), liquid chromatography (LC), and thin-layer chromatography (TLC).

5. Partition Coefficient

   • Defined as the ratio of concentrations of a solute in two different phases at equilibrium.

Equilibrium and Partition Coefficient Formula

The partition coefficient is defined as:
$K = \frac{\text{Conc. of solute in phase 1}}{\text{Conc. of solute in phase 2}}$

Types of Extraction Techniques

Methods of Extraction

• Batch Extraction: A process performed in batches.

• Continuous Extraction: An ongoing extraction method.

• Countercurrent Extraction: based on partition chromatography where two phases flow against each other.

Chromatography Terminology

• Solute: The substance to be separated.

• Stationary Phase: The phase on which separation occurs (can be liquid or solid).

• Mobile Phase: The solvent that carries the solute (eluting solvent, carrier gas, or supercritical fluid).

• Eluent: The substance that elutes the solute; to elute is to wash out the solute from the stationary phase.

• Chromatographic Peak: The graphical representation of the solute as it elutes from the column.

Chromatographic Principles

Chromatography utilizes the principles of partitioning or distribution of solutes between a stationary phase and a mobile phase, which is fundamental in food analysis.

Types of Chromatography Methods

Categories of Chromatography

• Liquid Chromatography: Includes normal-phase and reversed-phase techniques.

• Supercritical Fluid Chromatography: Uses supercritical solvents.

• Gas Chromatography: Involves the separation of volatile compounds.

• Thin-Layer Chromatography (TLC) and Paper Chromatography: Utilizes thin layers or paper to separate samples.

• Column Liquid Chromatography: Uses columns filled with stationary phase materials.

Characteristics of Chromatographic Methods

Paper Chromatography

Characteristics and Techniques

• Uses paper (cellulose) as a support for liquid stationary phase.

• The stationary phase is often water.

• A mobile phase can be a solvent that is immiscible with water.

Visualization Techniques

If the sample is colorless (e.g., certain amino acids), visualization can be achieved via:

• Autoradiography:

• Colorimetric methods: Such as ninhydrin, sulfuric acid, iodine vapor, dichlorofluorescein.

Rf Value Calculation

The Rf value of a component is calculated as:
$Rf = \frac{\text{Distance moved by component}}{\text{Distance moved by solvent}}$

• Note: The Rf values can be affected by various factors like stationary phase thickness, humidity, developing distance, and temperature.

Development Techniques

• 2-D Techniques: Mixtures can be developed in two dimensions by turning the paper and using different solvents for sequential separation.

Thin Layer Chromatography (TLC)

Features of TLC

• Developed to replace paper chromatography, offering better resolution due to smaller and more uniform particle sizes, speed, and reproducibility.

• Involves a thin layer of sorbent/bound stationary phase and uses various visualization approaches such as colorimetric methods and fluorescence.

Quantitative Evaluation

Quantitative evaluation can be performed via:

• Densitometry.

• Scraping off the zone, eluting the compound, and analyzing the resulting solution.

Column Liquid Chromatography

General Process

• Involves choosing appropriate stationary and mobile phases, along with column dimensions, isocratic vs. gradient phases.

Supercritical Fluid Chromatography (SFC)

Characteristics

• Utilizes a mobile phase commonly comprising liquid carbon dioxide, potentially mixed with methanol.

• Provides high resolution with low viscosity, suitable for non-polar and thermally labile compounds.

Ion-Exchange Chromatography

Interactions and Characteristics

• Selectivity based on ionic charge and size.

• Various types include anion-exchange and cation-exchange chromatography, with interactions primarily electrostatic in nature.

Ion-Exchange Mechanism

• Utilizes charged functional groups on stationary phases that interact with ions in the mobile phase.

Affinity Chromatography

Mechanism of Separation

• Based on reversible interactions between solutes and attached ligands on the stationary phase.

• Specific binding (e.g., with antibodies) allows for precise separation.

Elution Methods

• Nonspecific Elution: Involves changing conditions like pH or ionic strength.

• Bio-specific Elution: Uses excess ligand for elution.

Size-Exclusion Chromatography

Principle

• Molecules are separated based on their size, where larger molecules pass through more easily while smaller ones encounter hindrances due to available pore volume.

Key Terms

• Void Volume (Vo): Space occupied by the solvent around the particles.

• Elution Volume (Ve): Volume required to elute a given solute.

• Partition Coefficient (Kav): $Kav = \frac{Ve - Vo}{Vt - Vo}$ and describes the distribution of a solute between the mobile and stationary phases.

Analysis of Chromatographic Peaks

Key Concepts for Development

• Importance of knowing sample properties for separating distinct components.

• Optimization of methods, including chromatography type, stationary/mobile phase selection, and elution strategies (isocratic vs. gradient).

• Peak resolution (Rs): $Rs = \frac{2\Delta t}{w<em>1 + w</em>2}$ is crucial for differentiating peaks in analysis.

Quantitative and Qualitative Analysis

Quantitative Techniques

• Involves comparing peak heights or areas for concentration determination, using internal/external standards for calibration.

Laboratory Techniques

• Hands-on experience with HPLC, focusing on caffeine quantification and GC for profiling fatty acids.

Conclusion

The principles of chromatography play a crucial role in food analysis, providing various methodologies that can be optimized depending on the analysis undertaken. Understanding both qualitative and quantitative aspects enhances the effectiveness of chromatography in this field.