HPLC and LC-MS 2

Why are different scan modes used in mass spectrometry?

  • Electrospray ionisation (ESI) gives few fragment ions, so limited structural info

  • Mass spectrometry can be inefficient: ions of interest reach the detector for only a fraction of the scan time

  • Scan modes are used to:

    • Improve efficiency

    • Enable quantitation

    • Provide structural information even when fragmentation is minimal

🧪 Scan Modes in Mass Spectrometry

❓What are the main scan modes?

  • Product Ion Scan

  • Single Ion Recording/Monitoring (SIR or SIM)

  • Multiple Reaction Monitoring (MRM)

📊 Product Ion Scan

  • Select one precursor ion (usually molecular ion)

  • Fragment it in the collision cell

  • Second analyser scans all fragment ions

  • 🧠 Use case:

    • Useful with soft ionisation (e.g. ESI, CI) where fragmentation is minimal

    • Good for structural information

    • Can separate analyte from background ions

  • 📈 Diagram: [Show: Precursor ion → Collision Cell → Multiple fragment ions → Mass Analyzer]

🎯 SIR / SIM (Single Ion Recording / Monitoring)

  • Monitors a few selected ions (e.g., 4 ions in 1 sec scan = 0.25 sec per ion)

  • Significantly improves sensitivity (~100× over full scan)

  • 🧠 Use case:

    • Quantitation

    • Confirmation of known compounds

  • ⬇ If more ions are monitored → less time per ion → decreased sensitivity

  • 📈 Diagram: [Show: Only selected m/z values being recorded]

🎯 MRM (Multiple Reaction Monitoring)

  • Monitors a transition: specific precursor ion → specific fragment ion

  • Highly selective and sensitive

  • Requires prior characterisation of analyte

  • 🧠 Use case:

    • Quantitative analysis

    • Confirmation of compound identity

  • 📈 Diagram: [Show: Precursor → Fragment → Match both to confirm analyte]

🧾 Summary Table – Scan Modes

Scan Mode

Analyser 1

Analyser 2

Good For

Full Scan

All ions, sequential

–

RMM, some structural info

Product Ion Scan

One selected ion

All fragments

Structural info

SIR

Few selected ions

–

Quantitation, confirmation

MRM

Few selected ions

1–2 selected fragments

Quantitation, confirmation

🧬 Ionisation Techniques Summary

Technique

Ions Formed

Suitable for...

Fragmentation

EI

M+·

Small molecules

Yes

CI

[M+H]+

Small, polar

Sometimes

ESI

[M+nH]n+

Both small & large

Minimal

🔍 Examples:

  • Hexane (86 Da, non-polar) → EI

  • Proline (115 Da, polar) → CI

  • Haemoglobin (15,000 Da, polar) → ESI

💧 Ion Chromatography (IC)

❓What is ion chromatography used for?

  • Separation of ions and some polar molecules

📈 How does IC work?

  • Stationary phase: Contains ionic groups (e.g., NR₃⁺, SO₃⁻)

  • Mobile phase: Buffered aqueous solution with ions

  • Mechanism: Analyte ions compete with mobile phase ions for binding sites on the stationary phase

🔁 Ion Exchange Equation:

  • Cation Exchange: R-A⁺ + B⁺ ⇌ R-B⁺ + A⁺

  • A⁺: Weakly bound cation (mobile phase)

  • B⁺: Analyte ion

💡 Types of Ion Exchange

Type

Example Reaction

Anion Exchange

Resin-OH⁻ + H⁺Cl⁻ ⇌ Resin-Cl⁻ + H₂O

Cation Exchange

Resin-H⁺ + Na⁺HCO₃⁻ ⇌ Resin-Na⁺ + H₂CO₃

🧱 Stationary Phases

Type

Example Functional Group

Strongly Acidic

–SO₃H

Weakly Acidic

–COOH

Strongly Basic

–NR₃⁺

Weakly Basic

–NH₂

📉 Suppression in IC

  • Problem: Mobile phase contains ions → High background conductivity

  • Solution: Use a suppressor column to convert ions to weakly dissociated species

  • E.g., Carbonate → Carbonic acid

🚰 Suppression Reaction:

  • After Column: Bicarbonate buffer → Weak acid (H₂CO₃)

  • Reduces background → Better signal-to-noise ratio