Analysis Techniques

Electron impact is a straightforward ionization technique ideal for small, volatile molecules. The sample is vaporized and then bombarded by an electron beam with an energy of 70 electron volts, typically resulting in electron ejection and informative fragmentation patterns.

• Sample suitability: Best for compounds with a molecular weight below 400 Daltons, as larger molecules may thermally decompose during vaporization.
• Common applications: GC-MS for synthetic organic chemistry, hydrocarbon analysis, environmental water testing, and pharmaceutical or forensic drug screening.
• Sample preparation: Submit non-air-sensitive samples in an Eppendorf tube or small vial. Facility staff will transfer the material to specialized capillaries for analysis.
• Quantity requirements: Please provide between 50 micrograms (µg) and 1 milligram (mg) of material.

Chemical ionization is a “soft” ionization technique primarily used to enhance the abundance of the molecular ion. Similar to EI, it requires samples to be volatile and thermally stable. However, CI uses gas-phase ion-molecule reactions rather than direct electron bombardment to produce ions.

• Ionization process: A reagent gas (such as methane, isobutane, or ammonia) is ionized by electron impact. These reagent ions then react with sample molecules to produce stable, even-electron species, typically through protonation.
• Key advantages: Because the process is less energetic than EI, fragmentation is sparse. This significantly improves the likelihood of detecting the intact molecular ion (MH+).
• Fragmentation patterns: When fragmentation does occur, it usually involves the loss of small neutral molecules like water (H2O), ammonia (NH3), or acetic acid.
• Instrumentation: Our magnetic sector spectrometer is equipped with a CI source and integrated with an autosampler and gas chromatography (GC) system, allowing for complex mixture separation prior to analysis.

A typical reaction pathway using methane reagent gas is:

CH4 + e- → CH4+ + 2 e-

CH4+ + CH4 → CH5+ + CH3

CH5+ + M → CH4 + MH+ (protonation of the sample molecule, M)

Electrospray ionization is a versatile technique that generates gas-phase ions directly from solution (typically aqueous or organic solvents). By applying a high-voltage electric potential (typically 1.0 to 3.5 kV) to the liquid, the facility can analyze a wide range of polar and biological molecules.

• Mechanism: A fine spray of charged droplets forms at the capillary tip, creating a “Taylor cone.” As solvent evaporates, increasing charge density causes droplets to eject highly charged nanodroplets and unsolvated ions.
• Charge states: ESI uniquely produces multiply charged ions. While small molecules (<2000 Da) typically yield one to three charges, larger biomolecules can carry dozens of charges, allowing high-mass proteins to be measured within standard instrument m/z ranges.
• Applications: ESI is compatible with direct infusion or LC-MS and is ideal for lipids, metabolites, oligonucleotides, peptides, and proteins, as well as synthetic polymers and non-covalent complexes.
• Sample requirements: This technique is extremely sensitive to impurities, detergents, salts, and buffers. We recommend following our desalting protocol before submission.
• Solvent compatibility: Typical solvents include methanol, acetonitrile, and water. For non-typical solvents like DMF, chloroform, or THF, please consult facility staff.

The Synapt High Definition Ion Mobility Mass Spectrometer has an ion mobility analyzer which separates ions by ion mobility drift time. Unlike conventional mass spectrometers, the ion mobility analyzer can separate ions that overlap in the mass-to-charge ratio (m/z) dimension. The ability to orthogonally separate ions that overlap in the m/z dimension translates into a larger analytical peak capacity to provide more molecular identifications for complex samples that contain mixtures of compounds.

In addition, ion mobility spectrometry can reveal the presence of different conformers of a particular ion, which would be invisible to mass measurements alone. Understanding the higher order structure of biomolecules is important because different conformers may exhibit different chemical or biological activities.

New users must be trained by a facility staff member. Access to the MALDI room (B208B Stanley Hall) may be granted after the user completes a training session with a staff member.

New users interested in using MALDI mass spectrometry should contact Dr. Zhongrui Zhou.

The MALDI instruments are a Microflex LT/SH from Bruker and a Voyager DE Pro from Applied Biosystems.

Sample plates for the Microflex LT/SH can be obtained from Bruker. Compatible plates are: MSP 96-target ground steel BC, part number 8280799, and MSP 96-target polished steel BC, part number 8280800.

Sample plates for the Voyager DE Pro can be obtained from JBI Scientific. The most common plate is a stainless steel plate with room for 100 samples, part number V700666.

In matrix-assisted laser desorption/ionization (MALDI), the sample is embedded in a low molecular weight, ultraviolet-absorbing matrix that enhances desorption and ionization of the analyte. Sample preparation is crucial for successful MALDI. It is important to choose the correct matrix for your sample and to use suitable concentrations of both the sample and the matrix.

Commonly used MALDI matrices:

• α-cyano-4-hydroxy-cinnamic acid (CHCA)
  • Peptides, small proteins (<10 kDa), protein digests.
• 3,5-dimethoxy-4-hydroxycinnamic (Sinapinic acid)
  • Proteins (>10 kDa).
• 2,5-dihydroxy-benzoic acid (DHB)
  • Peptides, small proteins, small molecules, neutral carbohydrates, synthetic polymers, oligonucleotides (<10 bases).
• 2,4,6-trihydroxy-acetophenone (THAP)
  • Small oligonucleotides and acid-sensitive compounds.

This document offers tips on how to spot your sample.

And this document offers tips on how to remove buffers, salts, urea, guanidine, EDTA, glycerol, DMSO and detergents from your MALDI spots.

Each group is responsible for supplying their own sample plates, matrices, and other supplies necessary for sample preparation, such as micropipettes, microcentrifuge tubes and solvents.

Alternatively, you can contact Dr. Zhongrui Zhou to request assistance with analyzing your MALDI samples.