Liquid Chromatography: Key Components and Systems

Pumping Systems

• Requirements for HPLC Pumping Systems

  • Generation of pressures up to 6000 psi.
  • Pulse-free output to avoid irregular peaks.
  • Flow rates range from 0.1 to 10 mL/min.
  • Flow control and reproducibility of 0.5% relative.
  • Corrosion-resistant components to prevent damage and reaction with samples.

• Types of Pumping Systems

  1. Reciprocating Pump

     • Small chamber with back-and-forth piston motion.
     • Advantages: Small internal volumes (35-400 µL), high pressure outputs (up to 10,000 psi), constant flow rate, adaptable to gradient flow.
     • Disadvantages: Pulsed flow due to piston motion.

  2. Displacement Pump

     • Large syringe-like chamber with plunger driven by a screw mechanism.
     • Advantages: Flow is independent of viscosity and back pressure, pulse-free output.
     • Disadvantages: Limited solvent capacity.

  3. Pneumatic Pump

     • Collapsible container pressurized by compressed gas.
     • Advantages: Inexpensive, pulse-free.
     • Disadvantages: Limited solvent capacity and pressure output, dependent on solvent viscosity and back pressure, not suitable for gradient flow.

Sample Injection System

• Precision and Reproducibility

  • Sample introduced using a syringe into an interchangeable loop.
  • Supports pressures up to 7000 psi.
  • High precision with a few tenths of a percent RSD.
  • Addresses issues of reproducibility and overloading.

• Sample Sizes

  • Range from a few tenths of a microliter to 500 µL.
  • Interchangeable loops for different volumes.

Columns

• Types of Columns

  • Analytical Columns
    • Used for detection; lengths of 10-30 cm, inner diameter 4-10 mm, particle size 5-10 µm.
    • Common configuration: 25 cm length, 4.6 mm inner diameter, 5 µm particles.
    • Gives 40,000-60,000 plates per minute.
  • Guard Columns
    • Protect analytical columns by removing contaminants and preventing irreversible binding.
    • Made with similar composition but larger particle sizes.

• Column Packing Types

  • Particular Packing
    • Spherical, non-porous beads (30-40 µm) with surface coatings.
    • Used mainly for guard columns.
  • Porous Particle Packing
    • Particles 3-10 µm made of various materials, silica being the most common.
    • Used in liquid chromatography.

Detectors

• Bulk Property Detectors

  • Measure changes in solvent and solute properties (refractive index, dielectric constant, density).

• Solute Property Detectors

  • Measure compound concentration based on properties like UV absorbance, fluorescence, diffusion current.

• Detector Performance

  • Detectors vary in sensitivity (picograms, femtograms) and linear dynamic range.
  • Selection depends on property to analyze, detection limits, and available equipment.