chromatography and Mass spec

Chapter 6: Chromatography and Mass Spectrometry

Overview

• Focused on the principles and applications of chromatography and mass spectrometry in analytical chemistry and clinical laboratories.

Chromatography

Definition

• A group of techniques intended for the separation of complex mixtures based on different physical interactions.

Basic Components

• Mobile Phase: Can be a gas or liquid.

• Stationary Phase: Composed of solid or liquid.

• Column: Contains the stationary phase.

• Eluate: The separated components emerging from the column.

Chromatograms

• Instruments yield a response related to the amount of compound exiting the column, measured as a function of elution time or volume.

• The result is a chromatogram, a plot of response vs. time.

• Retention Time: Average time for a specific chemical to pass through the column. Increased retention times signify stronger interactions with the stationary phase.

Interpretation

• Chromatograms consist of various peaks, each representing different compounds.

  • Peak Area/Height: Indicates the quantity of the compound; sharper peaks imply better resolution and efficiency in separation.

Peak Performance

• Sharp Peaks: Indicate efficient separation, allowing simultaneous analysis of multiple compounds.

• Broader peaks can complicate measurements and negatively impact detection limits.

Factors Affecting Separation

• Column length, particle size, flow rate, viscosity of the mobile phase, and initial injection volume can all enhance chromatographic efficiency.

Modes of Separation

Types of Chromatography

• Adsorption Chromatography: Separation relies on interactions between the adsorbent (solid) and solute (liquid). Less common in clinical settings.

• Partition Chromatography: Based on solute solubility in two immiscible liquids (one polar, one non-polar).

• Ion-Exchange Chromatography: Uses ionic charge to separate solutes, beneficial for concentrating dilute samples and removing contaminants.

• Size-Exclusion Chromatography: Separates molecules based on size, mainly used for larger biomolecules like proteins.

High-Performance Liquid Chromatography (HPLC)

Characteristics

• Operates under high pressure, uses gradient elution for effective separation.

• Employs silica gel in columns and monitors eluate via detectors.

• Commonly used for quantitative analysis and DNA sequencing.

Gas Chromatography (GC)

Separation Technique

• Uses a carrier gas to transport compounds through a stationary phase within a column.

Advantages

• High resolution, low detection limits, and rapid analytical times.

• Pertinent for analyzing volatile substances, including therapeutic agents and environmental toxins.

Common Issues

• Sample injection issues include leaks or thermal decomposition affecting signal integrity.

Mass Spectrometry

Overview

• Technique for identifying unknown compounds, determining concentrations, and studying molecular structures.

• Key applications include clinical drug testing, identifying illegal substances in sports, and studying genetic disorders.

Mass Spectrometry Process

• Involves vaporization, ionization, and separation of compounds based on mass-to-charge ratios.

• Includes several ionization techniques:

  • Electron Ionization: Common in GC/MS.

  • Atmospheric Pressure Ionization: Used in LC/MS.

  • Electrospray Ionization: A technique that preserves molecular integrity.

Analyzing Results

• Mass spectrometry relies on three key components: ion source, mass analyzer, and ion detector.

• Detectors must offer high sensitivity, stability, and reproducibility across varied temperatures.

Clinical Applications

• Used for screening genetic disorders and assessing metabolic conditions in newborns.

• The Guthrie Test: Developed for early detection of phenylketonuria (PKU).

Applications of MS in Clinical Labs

• Key for structural determination, quantification of drugs of abuse, and detecting analytes with high specificity such as vitamins and hormones.

• Facilitates analysis of multiple analytes in one run, surpassing traditional techniques in sensitivity and reliability.