LC-MS Flashcards

Question

Components of a mass spectrometer (and roles)

Answer 1

Ionisation source (nebulisation, evaporation, ionisation) Ion focusing guides Mass analyser (m/z selection) Detector (m/z detection)

Answer 2

A technique used to produce ions at atmospheric pressure using an electrospray, in which a high voltage is applied to a liquid to create an aerosol.

Answer 3

1. Charge of ESI-capillary 2. Mist creation consisting of multiple charged droplets 3. Reducing surface/charge ratio leads to Coulomb explosion 4. Singly and multiple charged ions enter the MS

Answer 4

Ions are smoothly transferred from the atmosphere to the vacuum in several steps. To achieve this, a combination of several turbo molecular pumps (TMP) or of one multi-stage turbo pump together with a roughing pump is necessary.

Answer 5

Largely increases the probability of ions reaching the detector when there is a sufficient and stable vacuum.

Answer 6

Ions are transferred from atmosphere to vacuum Ions are focused and aligned with the help of electromagnetic fields Without ion optics only a tiny proportion of ions can reach the detector (<0.1%)

Answer 7

Separates ions of the same mass-to-charge ratio (m/z) from all other ions in a mixture using radio frequency (RF) Field voltage overlaid with direct current (DC) voltage

Answer 8

Q1 and Q3 voltages are optimised for a specific m/z precursor to product ion transition (precursor ion m/z > product ion m/z). MRM mode is commonly used in quantitative workflows as it allows for maximum selectivity and sensitivity

Answer 9

Separation of each mass from the next integer mass (the closest distinguishable separation between two peaks of equal height and width). Mass resolving power also depends on mass-to-charge ratio.

Answer 10

A metric describing the difference between the measured mass/charge (m/Q) of an ion and the real, exact m/Q of that ion. Mass accuracy depends on mass spectral signal-to-noise ratio and digital resolution. The measured m/Q is calculated based on the recorded time of flight and the input mass calibration.

Answer 11

A parameter commonly used to describe the width of a "bump" on a curve or function. It is given by the distance between points on the curve at which the function reaches half its maximum value.

Answer 12

Refers to the scans per second which can be performed per second. (rate at which you can acquire a mass spectrum (mass units/time))

Answer 13

It is apparent that anything that co-elutes with the analyte and affects the dynamics of the Coulombic explosion in any manner - for example, anything that affects the charge distribution or affects the surface tension of the droplet - will affect the detector response. These are collectively known as "matrix effects". Co-eluents will vary from sample to sample. Salts, fats, surfactants and other polar molecules are all perennial offenders. In LC-MSMS the instrument is tuned to only detect the analyte. Any co-eluting substance remains undetected, meaning that there is no warning that the signal in an individual sample may have suffered from a matrix effect and is any different than would otherwise be expected.

Answer 14

The time needed to acquire one spectrum (or one data point in a chromatogram)

Answer 15

- The acquisition of mass spectra in an orbitrap MS is based on the Fourier transformation of image currents and trapped ions - No magnetic field is involved - The ions perform an axial oscillation while rotating around a cylindrical inner electrode - The image currents are detected by the two outer electrodes - The mass spectra are generated by Fourier transformation of the time-domain signals into frequency-domain signals - The m/z value is inversely proportional to the square of the frequency - Orbitrap instruments allow ultra-high resolution measurements at relatively high speed - An important practical aspect to orbitrap MS performance is the adequate delivery of ions into the orbitrap. This is done in a pulsed way by means of a C-trap, which is essentially a curved high-pressure quadrupole capable of trapping ions and sending them as a concise ion package into the orbitrap.

Answer 16

As a stationary phase, PFPP shows better retention for small polar compounds, such as organic acids and amino acids, compared to traditional C18-based chromatography and is more stable in terms of retention time variation and required stabilization times, compared to HILIC

Answer 17

The highest confidence identification, a validated identification (Level 1), confirms a structure with a minimum of two independent and orthogonal data from a pure reference standard under identical analytical conditions.

Answer 18

A lack of reference standard acquisition but predictive or externally acquired structure evidence, namely MS/MS data, exhibiting diagnostic fragments or neutral losses consistent with a specific structure would be considered a putative identification (Level 2).

Answer 19

On the acceleration and collision of the selected ions with a target gas (He, N2, or Ar) in a collision cell, the ion translational energy can be partially converted into ion internal energy. If subsequent dissociation of the ion occurs in the collision cell, the process is called collision-induced dissociation.

Answer 20

CID is a two-step process: after converting ion translational energy into ion internal energy in an ultrafast collision event (∼10−15 s), unimolecular decomposition of the excited ions may yield various product ions by competing reaction pathways

Answer 21

Data-dependent acquisition (DDA) chooses which ions to fragment based upon intensities observed in MS1 survey scans and typically only fragments a small subset of the ions present.

Answer 22

A method of molecular structure determination in which all ions within a selected m/z range are fragmented and analyzed in a second stage of tandem mass spectrometry.

Answer 23

Dynamic exclusion temporarily puts the m/z of an ion into an exclusion list for a set length of time after its MS/MS has been acquired. As a result, the instrument will not continuously acquire MS/MS of the same ion, but spend more time acquiring MS/MS of less abundant ions.

Answer 24

This is the gas that assists with droplet formation. It helps to shear the droplets from the stream of liquid coming out of the inlet.

Answer 25

TEM is the temperature setting associated with the heating of the auxiliary gas. When nitrogen flows through, it gets heated and flows out as GS2, heating gas. The use of TEM and GS2 promotes desolvation and helps the ion evaporation process to ultimately condense the charged analyte, producing gas phase ions.

Answer 26

This is the voltage applied between the needle and orifice plate. The created strong electric field eventually pulls the formed ions into the analyzer.

Answer 27

The collision gas aids in fragmenting the precursor ions. When the precursor ions collide with the collision gas, they can dissociate to form product ions.

Answer 28

1. Nebulisation 2. Desolvation 3. Ionisation

Answer 29

Protonated/deprotonated molecules (adduct ions) are produced mainly by Brönsted-Lowry acid-base reactions or following the Lewis acid-base mechanism[5] or may be formed in the source itself. Eluent from the LC, carrying the ionized molecules, flows through a capillary probe containing a high-voltage electrode at the end of the flow path, and this draws ions of one specific polarity into each droplet, creating an excess. On the other end, a counter-voltage is applied to the sampling orifice electrode. The created strong electric field in between eventually pulls the ions into the analyzer, after the entire ion evaporation process. With the aforementioned assistance of nebulizer gas, charged droplets are easily formed.

Answer 30

The charged droplets are then attracted towards the sampling orifice. The size of the droplets will be reduced by a counter flow of heated nitrogen drying gas and uncharged material will be carried away by the drying gas as well. The charged droplets are then attracted towards the sampling orifice. The size of the droplets will be reduced by a counter flow of heated nitrogen drying gas and uncharged material will be carried away by the drying gas as well.

Answer 31

The desolvation increases the charge density on the surface of the smaller droplets, causing the increase of electric repulsion and resulting in droplet fission. When this process continues, the gas-phase ions are formed.

Answer 32

APCI is a gas-phase ionization technique. It is a process of evaporation followed by ionization, which is different from ESI. When the eluent flows out of a capillary, the LC solvent and analyte get quickly evaporated into gas phase in the heated vaporizer chamber. A corona discharge needle containing a small but potent electric current generates electrons and ionizes air and solvent molecules to form reactive species (e.g., H2O+, N2+, H3O+). The analyte then gets charged through charge transfer from the solvent reagent ions.

Answer 33

H = A + B/u + Cu equation to optimize plate height and linear velocity

Answer 34

QC samples provide a measure of the repeatability within an analytical batch and allow metabolic features with excessive drift in signal, retention time or accurate mass to be removed before data analysis in a QA process

Answer 35

Minimal height > maximum plates > greater separation

Answer 36

The tallest line in the stick diagram (in this case at m/z = 43) is called the base peak. This is usually given an arbitrary height of 100, and the height of everything else is measured relative to this. The base peak is the tallest peak because it represents the commonest fragment ion to be formed - either because there are several ways in which it could be produced during fragmentation of the parent ion, or because it is a particularly stable ion.

Answer 37

Because ions are very reactive and short-lived, their formation and manipulation must be conducted in a vacuum

Answer 38

Silica particles have surface silanol groups, SiOH. Silanol groups provide polar interaction sites. The reaction with monochlorosilane R(CH3 )2SiCl, where R =CH3 (CH2 )16-CH2 - will lead to "C18 silica" stationary phase.

Answer 39

The main advantage of the use of ESI for quantitative LC-MS is the formation of protonated or deprotonated molecules with little fragmentation, ideal for the selection of precursor ions and for maximising sensitivity. The major limitation is ion suppression or enhancement effects due to the presence of co-eluting analytes or co-eluting matrix components

Answer 40

Given that APCI is a gas-phase ionisation technique it tends not to suffer from ion suppression as much as ESI. However, considerable care is needed when selecting the type of solvent and additives used for the mobile phase to avoid issues with suppression of the analyte signal from preferential ionisation of solvents or additives with relatively higher proton affinities. APCI also needs greater concentrations of additives in the mobile phase to control the chemical ionisation process taking place in the gas-phase.

Answer 41

1. A small volume of your sample (in the liquid phase) is injected into your stationary phase. Your stationary phase is your HPLC column, which is a tube filled with particles <5µm in diameter. 2. A pump moves the liquid down the column using high pressure. As the sample moves through the column, there are interactions between the packing particles and molecules in your sample. This will cause these molecules to travel through the column at different rates. 3. As the individual components exit the column, a detector measures them. This output is sent to a computer to produce a liquid chromatogram.

Answer 42

Silica gel is by far the most common base packing material. The surface has silanol groups, which are highly polar and can interact with non-polar molecules in your liquid base. They also serve as chemical bonding sites and the large surface area offers a strong adsorptive capacity. Silica particles are rigid, which helps them to resist compaction. This is crucial when you’re using extremely high pressures for very small molecules. Low acidity silica is used to avoid interactions between bases and your liquid phase, improving peak shape.

Answer 43

For a given particle size, column efficiency will be directly proportional to the length of the column. However, pressure and elution time will also be directly proportional to the column length. Pressure and peak width will be directly proportional to the particle size for a given column length. Consider your sample and sample prep when choosing a particle size.

LC-MS Flashcards

(67 cards)