Mass Spectrometers
A chromatography technique that can let you separate different molecules in a sample based on its mass. This can be accomplished with different machines.
I google the difference between the two machines like clockwork, it's not even funny anymore so I know I need to write this down somewhere concrete. My lab is honing into the time where there may be more mass specs (3) than grad students in the lab (4).
Both machines in the lab are Liquid Chromatography Tandem Mass Spectrometers, as opposed to Gas Chromatography.
Triple Quad
First, what is a quad referring to?
Four parallel metal rods that can have voltages applied to it. As such, a triple quad has three of these quads, stylized as QqQ. Q1 has a direct current running through it and filters masses through it. Q2 is a collision cell that fires a specific voltage at the masses so it can fragment into a unique chemical fingerprint called Collision Induced Disassociation (CID) as a spectra. Q3 can select fragments of the masses to ensure you are really looking at what you think you are looking at. It can selection for various ionizations like H+
Guard Column/Column - the guard catches the messiest things first while the column interacts with the metabolites and affects retention time or how long the beads hold on to the metabolite. Particularly important to not clog the delicate downstream passages.
Orbitrap
Instead of using quadrapoles, the mass has an orbitrap where it is a barrel shape electrode rather than parallel rodes. It is able to trap ions as it spins around the barrel. Based on the spin, it is able to calculate the m/z through the angular frequency calculation: ω = √(k/(m/z)) -> k is the force constant. The vibration of the ion around the orbitrap is the angular frequency and thus, m/z can be calculated with precision.
Thermofisher Orbitraps are hooked up to a following Q to filter the mass.
Take note that the Orbitrab has better resolution, not sensitivity. It is able to determine the mass of molecules to the 5th decimal spot but it still "just okay" at detecting said molecules. Orbitraps have the benefit of using exact mass which makes them more useful for untargeted metabolite analysis. There is a large list of peaks that come out but the difficultly is determining which ones are important without a standard.
Order of processes
- Metabolite is prepared and injected into the machine and mixed with solvents and liquid nitrogen to move it through the capillary. Heat changes how they resolve.
- Nebulizer/Ion Spray - Converts the liquid sample into aerosol for detection
- Radio Frequency lens to narrow the ions down a tight channel and reduce overlapping noise.
- AABG - Beam guide to reduce noise or change the flux of metabolites
- Quadrapole mass filter
- Orbitrap mass analyzer
- HCD - Collision cell and detector to fragment different molecules to create a unique fingerprint.