Introduction to HPLC Chromatography

General Overview

• HPLC: High Performance Liquid Chromatography
• Chemistry Term: Chromatography
  • Separation of compounds
  • Visual separation of colors in early chromatography

Modern Chromatography Methods

• Gas Chromatography (GC): For volatile compounds with lower molecular weight
  • Operates at higher temperatures
• HPLC: For both volatile and non-volatile compounds
  • Only solubility is a concern
  • Less destructive; often run at room temperature

HPLC Mechanism

• Two Phases:
  1. Mobile Phase: Liquid solvent mixture (e.g., water and organic solvents)
     • Manipulation of polarity possible
     • Mobilizes compounds through the column
  2. Stationary Phase: Solid chemical phase inside the column
     • Various types (C18, C8, etc.)
     • Compounds adhere to this phase

Column and Separation

• Interaction: Between mobile and stationary phases determines separation
• Separation Process:
  • Sample loaded into column with mobile phase
  • Compounds adhere to stationary phase, then washed along
  • Separation based on polarity and affinity to phases

HPLC Components

• Mobile Phase Containers: Solvents for creating gradients
• Degasser: Removes air pockets from solvents
• Pumps: Ensure continuous flow of mobile phase
• Autosampler and Injector: Introduces sample into the column
• Detector: Identifies compounds and generates chromatogram

Chromatogram

• Retention Time (TR): Time a compound takes to travel through the column
• Dead Time (T0): Time non-retained materials take to exit
• Peak Area: Proportional to analyte amount; used for quantification

Types of HPLC

• Normal Phase: Polar column, non-polar mobile phase
• Reverse Phase: Non-polar column, polar mobile phase (e.g., C18)
• Ion Exchange: Columns for ion separation
• Size Exclusion: Separation based on size

Additional Points

• Choice of column and solvent affects separation efficiency
• Upcoming lectures will cover detailed HPLC types and troubleshooting
• Calibration curves for quantification in HPLC analysis

Lecture Conclusion

• Introduction to HPLC serves as a foundation for further study
• Future topics include column types, resolution improvement, troubleshooting
• Encourage engagement and support for the channel and website for additional resources

Note: These notes summarize the key concepts and mechanics of HPLC as discussed in the introductory lecture.