Lesson_20_-_Basic_Components_in_HPLC_system

Advanced Instrumental Analysis (LS5013FP) Lesson 20: HPLC Overview

Page 1

• Lesson: Basic components in HPLC system

Page 2

• Structure of the Lesson:

  • 1. Introduction

  • 2. Instrumentation of HPLC

  • 3. Safety precautions in HPLC laboratory

  • 4. Units in HPLC

  • 5. Summary

Page 3

• Lesson Objectives:

  • List the basic components in HPLC

  • Explain the functions of different components

  • State safety precautions when using HPLC

Page 4

• Introduction to HPLC:

  • HPLC can analyze larger compounds such as biological molecules and peptides.

  • Peptides and biological molecules are sensitive to heat, making HPLC more suitable than GC for their analysis.

Page 5

• HPLC Process:

  • Samples (liquid/solid) are dissolved in a solvent (mobile phase).

  • Separation occurs in the column through interactions between solute/stationary phase and solute/mobile phase.

  • Common detector used is UV/Vis.

Page 6

• Advantages and Disadvantages of HPLC:

  • Advantages:

    • High selectivity, suitable for non-volatile or thermally unstable compounds.

    • Accurate and allows for automation.

  • Disadvantages:

    • High cost and complexity.

Page 7

• Key Instrumentation Components in HPLC:

  • Injector

  • Column

  • Pump

  • Column oven

  • Data Processor

  • Detector

  • Degasser

  • Solvent Waste Reservoir

  • Reservoir

Page 8

• Instrumentation Breakdown:

  • 2.1 Mobile Phase

  • 2.2 Degasser

  • 2.3 Pump

  • 2.4 Injector

  • 2.5 Column Oven

  • 2.6 Column

  • 2.7 Detector

  • 2.8 Data Processor

Page 9

• Mobile Phase Details:

  • Solvents used as mobile phase and are stored in reservoirs.

Page 10

• Importance of Filtration:

  • Microfiltration (0.2μm to 0.45μm) of mobile phase is crucial to prevent clogs and ensure system integrity.

Page 11

• Solvent Polarity:

  • Variations in polarity affect elution and retention times of compounds.

  • Adjusting solvent percentage can modify the polarity.

Page 12

• Degasser Function:

  • Removes dissolved gases (nitrogen, oxygen) to avoid signal distortion and noise in detection.

Page 13

• Pump Role in HPLC:

  • Considered the heart of the HPLC system, driving mobile phase and samples to the column.

Page 14

• Injection Port Overview:

  • Injection volumes typically range from 10μL to 100μL.

  • Samples can be injected manually or using an automatic sampling loop.

Page 15

• Column Oven Functionality:

  • Temperature affects separation; increased temperature typically decreases retention times.

  • Optimal analysis temperature is around 35°C.

Page 16

• Safety Precautions in HPLC Lab:

  • Health Risks:

    • Toxic solvents, pulmonary irritation from stationary phase, and high pressure hazards.

Page 17

• Managing Toxic Solvents:

  • Long-term exposure can cause health issues.

  • Implement good laboratory practices (GLP) to mitigate risks, such as using covered containers and providing good ventilation.

Page 18

• Safety Cap Use:

  • Safety caps prevent solvent vapors by sealing containers, only allowing airflow during pump operation.

Page 19

• Particle Risks:

  • Small particles from stationary phase can enter the lungs; operations should be conducted in a fume cupboard.

Page 20

• Units of Measurement in HPLC:

  • Pressure: psia, psig; 1 bar = 0.987 atm, 105 Pa, 14.5 lb/in².

  • High-pressure capabilities of HPLC pumps can reach up to 500 bar.

Page 21

• Length Measurements:

  • Conversion: 1m = 1,000mm = 1,000,000μm.

  • Example: Diameter of human hair is about 50-100μm, comparing sizes for understanding.

Page 22

• Summary of HPLC:

  • HPLC is a separation technique where sample mixtures interact with stationary and mobile phases, allowing for separation based on component affinities.

Page 23

• Mobile Phase Transport:

  • Mobile phase carries sample components through the column.

  • The choice of mobile phase depends on sample characteristics.

Page 24

• Pumping and Injection Summary:

  • Pumps transport mobile phases at specific flow rates; injections are executed via a loop system, and a column oven maintains consistent temperature during analysis.