lecture 6- liquid chromatography mass spectrometry

LC-MS work flow

• HPLC System

  • Uses an autosampler for sample injection and an isocratic pump for consistent solvent flow.

  • Multiple detectors can be added to enhance analysis.

• LC to MS Interface

  • The interface transfers separated compounds from the LC system to the MS.

  • The interface also acts as the ionization technique (e.g., ESI or APCI).

• Mass Spectrometry

  • Ions are analysed based on their mass-to-charge ratio (m/z) to identify and quantify analytes.

LC-MS

• Separates complex mixtures using liquid chromatography (LC).

• Ionizes compounds for detection using mass spectrometry (MS).

• Provides structural and molecular information for each component.

• Produces mass spectra for detailed analysis of each chromatographic peak.

LC-MS columns

• Narrow Bore:

  • Reduces sample dispersion, improving resolution.

• Lower Particle Size:

  • Increases surface area for separation, enhancing separation efficiency and resolution.

• Short, High-Resolution Columns:

  • Provides high throughput and efficient separation while maintaining high resolution for accurate analysis.

LC-MS ionisation techniques

- electrospray ionisation

- atmospheric pressure ionisation

- atmospheric pressure photo ionisation

electrospray ionisation

• Sample Introduction:

  • Sample is introduced as a liquid phase through a fine needle or capillary.

• High Voltage Application:

  • A voltage (typically 3-5 kV) is applied to the needle.

• Formation of Charged Droplets:

  • The voltage creates electrical stress between the needle tip and a counter electrode.

  • This forms highly charged droplets as the solvent is sprayed.

• Solvent Evaporation:

  • Nebulizer gas helps to evaporate the solvent, causing the droplets to shrink.

• Ion Formation:

  • As the droplets shrink, the ions become more concentrated, leading to the formation of charged analyte ions.

• Transition to Gas Phase:

  • The charged ions transition from liquid to gas phase, ready for analysis by mass spectrometry.

• Soft Ionization:

  • Minimal fragmentation of the analyte occurs, preserving its structure for further analysis.

advantages of ESI

• Simple ionization of non-volatile solutions.

• Easily combined with chromatographic systems (e.g., LC).

• Multiple ionization modes available (positive/negative).

• Provides accurate molecular mass and structural information.

disadvantages of ESI

• Careful optimization of experimental parameters needed.

• Limited solvent/solution choices.

• Fluctuating ion signal can affect stability.

atmospheric pressure chemical ionisation

• Creates ions at atmospheric pressure.

• Sample is heated, volatilized, and nebulized with nitrogen gas.

• Ionization occurs in the gas phase by a corona discharge.

• Best for polar, semi-volatile samples.

• Supports both positive and negative ionization modes.

advantages of ACPI

• Direct observation of the molecular mass spectrum.

• Handles higher flow rates compared to Electrospray Ionization (ESI).

• Ideal for weakly polar and semi-volatile compounds.

• Good sensitivity and robustness for small to medium-sized molecules.

• Supports both positive and negative ion modes.

disadvantages of ACPI

• Not suitable for biological macromolecules (cannot generate multi-charged ions).

• Limited structural information (produces few fragment ions).

atmospheric pressure photo ionisation

• Sample is nebulized and vaporized with a heated nitrogen gas stream.

• Ionization occurs by absorption of UV photons.

• Works best with non-polar and less polar compounds.

• Can operate in both positive and negative ionization modes.

Advantages of APPI

• Good for non-polar and weakly polar compounds.

• Lower chemical noise compared to APCI.

Disadvantages of APPI

• Less effective for highly polar compounds.

• UV lamp degradation over time requires maintenance.

mass analysers for LC-MS

- quadrupole

- time of flight

- ion trap

- orbital trap

time of flight mass analyser

• Ions are accelerated to the same kinetic energy.

• Lighter ions reach the detector faster than heavier ions.

time of flight mass analyser advantages

• Very fast analysis.

• High mass accuracy and resolution.

• Wide mass range good for small and large molecules.

ion trap analyser

• Traps ions in a 3D electric field.

• Sequentially ejects ions based on mass-to-charge ratio (m/z) for detection.

• Can isolate, fragment, and analyze ions within the trap (MS/MS capability).

ion trap analyser advantages

• High Sensitivity: Detects low-abundance ions effectively.

• Good Resolution: High mass resolution for detailed analysis.

• Compact & Inexpensive: Smaller and cheaper compared to other MS types.

ion trap analyser disadvantages

Limited Dynamic Range: Struggles with detecting very low and very high-abundance ions simultaneously.

Lower Sensitivity for Complex Samples: Less effective with highly complex mixtures.

orbital trap mass analyser

• Ions are injected into a central spindle electrode, where they oscillate in a static electric field.

• Their oscillation frequency is linked to their mass-to-charge ratio (m/z).

• The data is converted into a mass spectrum

orbital trap mass analyser advantages

• High Resolution & Accurate Mass

• No Magnetic Field required

• High Sensitivity for low-abundance ions

• Good for Complex Samples

orbital trap mass analyser disadvantages

• Slower Speed compared to other MS types

• Expensive technology

• Requires Complex Maintenance

• Limited Dynamic Range

tandem MS

two or more mass analyzers

• Ion Formation:

  • Ions are formed in the ion source and enter the first mass analyzer.

• Fragmentation (CID):

  • Ions are then fragmented by collision-induced dissociation (CID) in the collision cell, where ions collide with an inert gas, breaking them into smaller product ions.

• Detection of Product Ions:

  • The second mass analyzer detects the product ions, allowing for detailed structural or compositional analysis.

Advantages of tandem MS

• Improved Sensitivity:

  • Better detection of low-abundance ions in complex samples.

• Better Resolution:

  • Helps isolate and analyze ions with similar m/z ratios.

• Structural Information:

  • CID provides detailed insights into the structure of molecules by analyzing their fragments.

• Ideal for Complex Samples:

  • Useful in proteomics, metabolomics, and other applications requiring high precision.