Lecture 15: Mass Spectrometry

What can mass spec help us do?

Information gained:

Elemental composition of matter

Structure of inorganic, organic and biological molecules

Qualitative and quantitative composition of complex mixtures

Structure and composition of solid surfaces

Isotopic ratios of atoms in sample

Application for routine chemical analysis since early 1940s

Identifying structures in organic chemistry

Possible to produce ions from non-volatile or thermally labile molecules

What questions does mass spec answer?

What’s in a certain sample?

What is the structure of the molecule

How much of the substance can be quantified?

Mass Spectrometry Compounds

mass spec determines the mass of molecules by measure the mass-to-charge (m/z) ratio of its ions

ions are generated by inducing a loss or gain of a charge from a neural species

electrostatically directed unto a mass analyzed and separated acordibg to m/z ration

ion source → where the ions are made; gas charged molecules

** can be inside the vaccum or outside (when outside, it is called atmospheric ionization

mass analyzer → really a mass to charge analyzer

Sample Introduction

critical step

multiple possible techniques → direct infusion, gas chromatography, liquid chromatography, and electrospray ionization

method of choice depends on the samples physical state, chemical properties,and desired analysis

the method of introduction can influence ionization efficiency, detection limits, and overall performance of the mass spectrometer

Ion Sources

• Can be through soft or hard ionization

• Ionization methods:

  • Electron Ionization (EI) → energetic electrons

  • Matrix-assisted desorption/ionization (MALDI) → laser beam

  • Fats atom bombardment (FAB) → energetic atomic beam

  • Electrospray Ionization (ESI) → high electrical field

hard ionization

fragment the molecules

soft ionization

keep the molecules intact

Parts of Electron Ionization

Electron Source: Generates high-energy electron,s, typically from a heated filament

Ionization Chamber: Where the sample molecules are bombarded with electrons

Sample Inlet: Designed for vapor introduction, often coupled with gas chromatography

Electron multiplier: commonly used for detecting resulting ions

Quadrupole Mass Analyzer: often used for mass analysis after ionization

Characteristics of Electron Ionizzation

High Energy Process: Involves bombarding vaporized molecules with high-energy electrons, typically 70 eV causing ionization and fragmentation

Fragmentation Patterns: Procedures use distinctive fragmentation patterns for structural elucidation and compound identification, especially in organic molecules

Primary Volatile Compounds: Best suited for small, volatile, and thermally stable compounds

Common in GC-MS: Frequcntly used in combination with Gas Chromatoigraphy (GC) for analyzing gaseous and volatile compounds.

Universla Detector: Considered a ‘universal’ ionization method due to its effectiveness across a broad range of compounds

Electrospray ionization

• An atmospheric-pressure ionization method allowing the transfer of dissolved macromolecules into a MS by converting them into ions via the gas phase

• Used for analysis of non-volatile compounds

• Can use low concentrations of analyte

• Forms multiple charged ions by forming multiple adducts with the analytes

• Allows for coupling of LC or CE to MS

How is Electrospray Ionization a form of soft ionization

Spray Capillary: Delivers the liquid sample and creates and aerosol

High Voltage Source: Applied to the spray capillary to induce ionization

Heated Voltage Source: Applied to the spray capillary to induce ionization

Heated Capillary and Desolvation Chamber: Used to evaporate solvent and focus ions

Ion Optics: Directs the ions from the spray source inro the mass analyzer

Mas Analyzer: Often a Time of flight, quadrupole or orbitrap for mass analysis0

Soft Ionization: Matrix Assisted Laser Desorption/Ionization (Maldi)

• A sample is mixed with a matrix, most often simple organic solvents

• The mixture is dried on a sample plate and ablated with a laser beam to create a plume of analyte and matrix ions

• Matrix ions transfer charge to analyte

• generally uses a UV laser, IR is also possible (not common) ions are formed by gas-phase proton transfer between excited matrix ions and the analyte

***generally, only single charges are observed

Resolution and mass analyzers

• separates ions based on their mass to charge ration

• It has very good mass accuracy and resolving power (resolution) along with measurement speed

• Resolution m/Δm

• “M” is the m/z of the first peak and

• “Δm” is the distance between two peaks

Mass Analyzers: Quadrupoles

• Most common and contains four poles, electric voltage is applied fluctuating and usually in pairs

• The ions oscillate through the electric field only a select mass range can make it to the detector

• Compact and less expensive

• Short scan time

• Varied DC source to bias rods and accelerate ions

• Oscillating RF voltage signal on top of the DC bias, occasionally flip polarity

Quadrupole Ion Trap

End-cap electrodes are held at ground separated by ring electrode with a fixed frequency RF applied

Time-of-Flight Mass Analyzers

When ions have the same kinetic energy, the smaller m/z ion flies faster than the larger m/z ion

Measuring the flight time of ions can provide a mass spectrum

R = m/Δm

Tandem Mass Spectrometry

Mass Spec experiments can illustrate structural information about the analyte using precursor ions to break down into product ions, which reveal structural data about the precursor ions

MS v. MSxMS

MS → tells you the mass/elemental composition of the compound

MS x MS → tells you the structure of the compound (certain functional groups w. known masses may break off, may help us determine the structure)

How does the quadrupole act as a mass filter?

 We're getting rid of the rest of the sample and measuring one m/z ratio. 

The molecules that are too big or small fly to the rods and are destroyed because they can't maintain that orbit in the rods.  

Block Diagram of a Mass Spectrometer

Which ionization methods are hard and which ones are soft

Hard:

Electron Ionization - the molecules are being bombarded w electrons, causing a lot of fragmentation. Because of this you are more likely to have a fragment of a full molecule

Soft

Electrospray Ionization - soft because you aren't charging the analyte directly, but instead the mobile phase and the matrix

MALDI - soft because the sample is mixed with the matrix if it was just the sample, it would've have been destroyed by the laser

How does Electrospray ionization work?

needle charges droplets. Hot gas is blown on to the solvent, so it evaporates. The droplets get smaller, and the charge density gets closer together, causes a coulombic explosion that transfers from the solvent to the analyte

False; They create singly positively charged ions 

True or False: Electron Ionization can only produce singly and negatively charged ions.

True

E. True or False: An electron multiplier tube is more sensitive and has a lower limit of detection than a Faraday cup. 

Faraday Cup

standard detector for mass spec

1. metal cup connected to a circuit

2. molecules flies into metal cup and trasnfers charge

3. resulting current from the cup is the signal measured

False;  ESI uses hot gas, and MALDI is usually atmospheric. 

F. True or False: All ionization methods require a vacuum to operate effectively. 

True;  Quadrupole, Time of Flight, and Ion Trap all require vacuums. 

G. True or False: All mass analyzers require a vacuum to operate effectively. 

Quadrupole and time of flight

A (quadrupole / time of flight / ion trap) can only separate out one m/z ratio at a time, requiring it to “scan” through the entire sample. 

How do quadrupole mass analyzers work?

four poles, ions fly through as the em field changes, only one molecule can pass through at a time

Scan through mass to charge ratios for one molecule over a couple seconds

How do time of flight mass analyzers work

you have all of the ions in an electric tube, you give them all one push with the same KE. The objects w less mass will fly faster through the drift tube

How do Ion Trap mass analyzers work

• 4 metal plates creating an em field

• your ions are going to stay in orbit in that magnetic field when you change the em field one at a time, you are kicking out ions that are in the trap

• you are destabilizing the orbit

all of them do. the whole point of mass spec is that we are manipulating the em field to separate molecules by mass

this is why every measured molecule has be an ion

A (quadrupole / time of flight / ion trap) uses electromagnetic fields to accelerate ions

how is the fragmentation in mass spec reproducible