Final Exam: Mass Spectrometry

Be able to explain how a linear TOF detects ions. What are the advantages and disadvantages?

- ions formed in the source are accelerated through a high potential where they acquire the same kinetic energy - then they enter a field-free region where they separate because different ions of different m/z values have different velocities - ions are separated in the flight tube based on differences in their velocities - the flight time of an ion is proportional to length of the flight tube, the accelerating voltage and its mass

How is detection done differently on Orbitrap and FT-ICR mass spectrometers

- magnetic field traps ion - can excite orbit and change voltage frequency on the plate - ion absorbs energy, orbit expands, frequency stays the same - frequency proportional to mass of the ion

Be able to describe how electron ionization is carried out

- occurs through interaction of the gas-phase sample molecules with high-energy electrons emitted from a resistively heated filament - After emission from the filament, the electrons are focused through a narrow slit and attracted to an anode that is positioned ~15-20 mm opposite the filament.

How are unit and accurate mass measurements different?

- accurate mass measurements: involve measuring ion masses to one thousandth or ten-thousandth of a Dalton - unit mass measurement: involve measuring ion masses to a single dalton

Be able to describe the advantages and disadvantages of Multichannel Plate detector for mass spectrometry. Which mass analyzer is this paired with?

- advantage: improved time resolution and circular with diameters up to several centimeters - disadvantage: the moderate magnetic flux densities prevent electrons from hitting the tube walls; gain not as good - paired with time of flight mass analyzers

Be able to describe the advantages and disadvantages of channeltron detector for mass spectrometry. Which mass analyzer is this paired with?

- advantage: the electrons dislodged by the incoming ion will strike farther down the surface where the voltage is higher, creating more electrons - paired with quadrupole

Be able to describe the advantages and disadvantages of photomultiplier detector for mass spectrometry. Which mass analyzer is this paired with?

- advantages: long life relative to other detectors and gain in signal for each ion that strikes the detector - disadvantages: mechanically fragile - paired with quadrupole

Be able to describe how an ion trap mass spectrometer traps and detects ions

- analyze ions formed in the source of a mass spectrometer by first trapping them in a three dimensional space and then ejecting them one m/z value at a time to detector to record a mass spectrum - consist of two endcap electrodes and one ring electrode - an electrostatic ion gate pulses open (-V) and closed (+V) to inject ions into the ion trap as they flow out of an ion source - ions formed in the source of a quadrupole are lost if the voltages on the four rods are not configured to pass them to the detector

Be able to explain how a single quadrupole mass analyzer selects ions for analysis

- comprise four parallel rods of circular or hyperbolic cross-section - capable of unit mass analysis with a few exceptions - ions formed in the source are accelerated in an electric field into the mass analyzer where ions having different m/z values are selected to pass to an ion detector based on their interactions with electric fields applied perpendicular to their flight path - the acceleration voltage in the source is constant but the electric fields applied in the x and y directions by circular rods will vary

Be able to describe how electrospray ionization is carried out. Know how to calculate the molecular weight of a protein from an electrospray mass spectrum.

- electrospray is the ionization method that is coupled with liquid chromatography - a technique that applies a high voltage to a liquid to produce an aerosol - Due to relatively fragile biomacromolecules, their structures are easily destroyed during the process of dissociation and ionization Calculate molecular weight: m/z=(Molecular weight + H+)/(H+)

Be able to explain how a reflectron TOF detects ions. What are the advantages and disadvantages?

- has increased mass resolution compared to a linear TOFMS because ions of different m/z values travel greater distances relative to a linear instrument - composed of one field-free region followed by a region with a reflecting potential mirror, which is followed by another field-free region, it enters a reflector region where a potential with the same polarity of the ion is applied - this potential effectively slows the ion down to zero velocity and then reaccelerates out fo the reflector region into the second field free region - more energetic ions will penetrate the electric field a greater distance before reversing their flight path - this has the net effect of making ions with slightly larger thermal velocities travel a greater distance, which narrows the flight time difference between ions of the same m/z values with different initial thermal energies, thus increasing mass resolution

How do ion traps compare to single and triple quadrupoles in terms of experiments performed and relative sensitivity?

- ion trap mass analyzer will perform all the same experiments a triple quadrupole mass analyzer will (full scan, product ion scans, SIM, and SRM determinations) - sensitivity gains going from full scan mode to SIM or SRM mode using an ion trap

How do MS/MS experiments (product ion and single reaction monitoring) carried out on a Q-TOF compare with a triple quadrupole mass spectrometer?

- overall sensitivity for the product ion scan is better than a triple quadrupole as well because TOFs do not need to scan over individual m/z values - major advantage of the triple quadrupole vs. a Q-TOF is the sensitivity with which one can perform single reaction monitoring experiments used as selective detection method in LC/MS/MS analyses of complex mixtures

Be able to describe the mechanism of MALDI mass spectrometry

- prepared by mixing or coating with solution of an energy-absorbent, organic compound called matrix - When the matrix crystallizes on drying, the sample entrapped within the matrix also co-crystallizes - The sample within the matrix is ionized in an automated mode with a laser beam

Be able to describe product ion analyses, as well as single (and multiple) reaction monitoring experiments, are carried out. What information do you get?

- product ion: carried to acquire structural information from a single m/z value exiting the source - first quadrupole is used to select a single m/z value to pass into the second quadrupole (Q1) which is a collision cell - The ion selected by the first quadrupole then is made to undergo fragmentation into smaller ions in the collision cell (Q2) by collisions with an inert gas - some of the translational energy of the ion is converted to internal energy during the collision, and that newly acquired internal energy causes bond cleavage leading to the formation of other fragment ions - the third quadrupole (Q3) then undergoes full scan over a predetermined range to detect the fragments formed in the Q2 collision cell - single and multiple reaction are carried out same way - information get: range of m/z and molecular weight

Be able to explain how a triple quadrupole is configured. What information do you get?

- system with an ion source, three single quadrupole mass analyzers in tandem, and an ion detector - can be configured to do SIM and full scan analysis like a single quadrupole - may also be used to do product ion analysis and single reaction monitoring

Be able to describe a Q-TOF mass spectrometer

- the combination of the two mass analyzers allows one to acquire product ion data under accurate mass conditions, which is not possible on a triple quadrupole mass analyzer - the quadrupole mass analyzer can be used to select an ion with a specific m/z value that passes into a collision cell where it fragments by collisions with an inert gas - all the products ion scan be analyzed at the same time using a TOF mass analyzer

What are the two common experiments done with a single quadrupole mass analyzer? How is the voltage changed in each of these experiments?

1. full scan: which is done to answer questions concerning the molecular weight, empirical formula, and primary structural features of an unknown molecule 2. Single ion monitoring: only select m/z values are monitored over a predetermined range of retention times. SIM is used for quantification - change voltage as function of time to detect different ions