HPLC and LC-MS Overview

Liquid Chromatography (LC / HPLC)

• Used for separating compounds that are:

  • Small or large

  • Polar

  • Thermally unstable or stable

  • Involatile

• Key components:

  • Mobile phase

  • Stationary phase

  • Detector

🧪 High Performance Liquid Chromatography (HPLC)

• Types of HPLC:

  • Normal phase: The stationary phase is more polar than the mobile phase

  • Reverse phase: The stationary phase is less polar than the mobile phase

• The vast majority of HPLC experiments are conducted using reverse phase systems.

🌱 Normal Phase HPLC

• Stationary phase: Polar materials such as silica or alumina

• Mobile phase: Non-polar solvents like hexane or dichloromethane

• Retention behaviour: More polar analytes are retained longer due to stronger interaction with the stationary phase

🌊 Reverse Phase HPLC

• Stationary phase: Non-polar (e.g., alkyl-modified silica like C18, C8, or C4)

• Mobile phase: Polar solvents such as water, methanol, or acetonitrile

• Retention behaviour: More hydrophobic analytes are retained longer

🔍 Key Considerations in HPLC Design

Stationary Phase

• Particle size:

  • Smaller particles → Higher surface area → Better resolution

  • But also → Requires higher pressure

• Pore size:

  • For molecules <3,000 Da → Use ≤100 Å pores

  • For 3,000–10,000 Da → Use 100–130 Å pores

  • For >10,000 Da (e.g. peptides/proteins) → Use 300 Å pores

Column Dimensions

• Affects sensitivity and loading capacity:

  • Larger columns → Good for preparative HPLC

  • Standard columns → Analytical use

  • Small diameter columns → Higher sensitivity but lower sample capacity

Flow Rate

• Higher flow rate → Shorter retention time

  • Pros: Faster analysis

  • Cons: Higher solvent use, potentially reduced resolution

Elution Method

• Isocratic elution: Mobile phase composition remains constant

• Gradient elution: Mobile phase composition changes during the run (e.g., increasing organic content in reverse phase HPLC)

📊 Quantitative Analysis in HPLC

• Peak area is directly proportional to the amount of analyte injected

• Peak height is less reliable due to potential peak broadening or asymmetry

Calibration Methods

• External calibration:

  • Standards are run separately from samples

  • Assumes identical conditions for all runs

• Internal standard calibration:

  • A standard is added to both samples and calibration solutions

  • Compensates for injection or detection variability

🔎 Detectors in HPLC

UV Detectors

• Require chromophores to detect compounds

• Derivatisation methods:

  • Dansylation for amino acids

  • Phenylhydrazine for ketones

  • 3,5-dinitrobenzoyl chloride for alcohols

• Types of UV detectors:

  • Dispersive: Records one wavelength

  • Diode Array (DAD): Records full spectra or multiple wavelengths

• UV absorbance by solvents can be problematic – HPLC-grade solvents are used to minimise this.

⚡ HPLC-MS (LC-MS)

• Ionisation technique: Electrospray Ionisation (ESI)

  • Soft ionisation method suitable for thermally labile and high molecular weight compounds

  • Produces multiply charged ions, allowing detection of large molecules within the m/z range of mass spectrometers

• Example:

  • Protein of 20,000 Da, with 20 added protons:

    • Mass = 20,020

    • Charge = 20

    • m/z = 1,001 (within measurable range)

⚙ Mass Analysers

• Function: Separate ions based on mass-to-charge ratio (m/z)

Types of Analysers

• Low resolution: Quadrupole, Ion trap

• High resolution: Time-of-flight (ToF), Magnetic sector, FT-ICR

Key Properties

• Resolution (FWHM): How well two nearby peaks can be separated

• Mass accuracy: How close the measured mass is to the actual value

Use of Accurate Mass

• Identifies elemental composition

• Example:

  • CO = 27.9949

  • N₂ = 28.0062

  • Distinguishable by high-res MS due to non-integer relative atomic masses

Tandem MS (MS/MS) Configurations

• Triple Quadrupole (QqQ)

• Quadrupole-Time of Flight (Q-ToF)

• Time of Flight – Time of Flight (ToF-ToF)