hyphenated techniques

what is nominal mass?

integer mass of ion made up of lightest stable isotopes of each element

what is exact monoisotopic mass?

mass of ion that is made of lights stable isotopes of each element

what is average mass?

mass of ion calculated using relative average isotopic mass of each element

what is isotopic abundance?

naturally occurring distribution of same element with different atomic mass

what are isobaric mass empirical formulae?

same nominal mass gut different exact monoisotopic masses

how is exact monoisotopic molecular mass calculated?

remember to subtract mass of electron

how to calculate intensity of M+1/intensity of M peak?

natural abundance of each +1 isotope (e.g 13C) x exact mass / lightest stable isotope x natural abundance

what does a full scan give? what type of analysis? 2D or 3D why?

maximum info

large volumes of data

don’t visualise as 3D as too complex

good for non targeted analysis

what is a total ion chromatogram?

integrates signal over all m/z values

what is a base peak chromatogram?

most intense m/z peak at each time point

what is extracted ion chromatogram?

collects full scan and extracts data for specific m/z

what is selected ion monitoring? advantages? what type of analysis?

only collect at specific m/z (don’t collect full scan)

collect many more data points = smoother chromatogram, less data generated

used for targeted analysis

quadrupole MS

what is the principle of mass analysis? low or high res

filters ions by oscillating electric fields

low res

triple quadrupole MS (QqQ)

what is the principle of mass analysis?

low or high res

two quadrupoles + collision cell for MS/MS

low res

time of flight MS

what does it measure? high/low res?

measures time ions take to reach detector

high res

quadrupole time of flight MS

how does it work? low or high res

quadrupole for selection

time of flight for detection

high res

GC-MS

how do analytes leave the column?why?

what are the sources of ions?2

leave column at low conc in high temp gas = suitable for introducing into mass spec after species ionised

ions from electron impact or chemical ionisation

label this electron impact quadrupole mass separator

• where does sample come from

• how does sample become ionised, where?

  • what happens to electrons?

• how does mass analyser work? what determines whether or not the ion reaches detector?

constant voltage and radio freq alternating voltage

causes electrons to spiral through 4 rods. radio freq determines which electrons reach the detector - others collide with quads and are lost

time of flight mass separator

why is tube bent?

how does the reflection detector improve resolution?

ions have same m/z but different kinetic energy depending on where they are

tube is bent so they arrive together, otherwise would arrive at different times

refocuses accelerated ions

where is electron lost from in electron impact? (oxygen)

HOMO - non bonding orbital centred on O or lone pair

show general steps of ms/ms mass analysers (how many are there?), show how sample changes form and is sorted

what is selective reaction monitoring?

measures peak due to a specific daughter ion of a specific parent ion

• even more selective and less noise

what are the two things a mass analyser could do to only allow specific m/z through

selective reaction monitoring

selecting parent ion and scan spectrum of daughter ions

show triple quadrupole for GC-EI / LC-ESI?

show how parent ions become daughter ions, and how the mass separators sort the parent ions, and then the daughter ions

how are all m/z signals collected in turn?

for these general steps

product ion scan - what is allowed through Q1? what is q2? what does Q3 do?

what is chromatogram and mass spec and what does it show?

only particular m/z ratio allowed through Q1

collision induced dissociation for q2

all allowed through Q3

• chromatogram - peaks due to compounds with precursor ion

• all fragment ions of precursor ions

for these general steps

precursor ion scan - what is allowed through Q1? what is q2? what does Q3 do?

what is chromatogram and mass spec and what does it show?

Q1 - all allowed through

collision induced dissociation for q2

only particular fragment allowed through mass separator for Q3

• chromatogram - peaks due to all compounds giving product ion

• all precursor ions with same product ion

for these general steps

selective reaction monitoring - what is allowed through Q1? what is q2? what does Q3 do?

what is chromatogram and mass spec and what does it show?

Q1 - one m/z ratio allowed through

collision induced dissociation for q2

only one fragment allowed through Q3

• chromatogram = peaks due to specific compounds with specific precursor/product ion combo

• precursor/product ion pair being monitored

what must the analyte be for GC? 2

what does derivatization increase? how?

analyte must be volatile and thermally stable

increases volatility - replace polar groups with non polar to lower BP and increase vapour pressure

how could you make this less polar to increase volatility?

replace COOH with methyl ester group

how could you make this less polar to increase volatility?

can no longer form H-bonds

how could you make this less polar to increase volatility?

• silylation

why are silyl are fluoroalkyl compounds more volatile than alkyl ones?

intermolecular forces are weakened

what state must instrument sample be when injected for GC, GC-MS or GC-MS/MS?

gas or solution of analyte in volatile solvent that isn’t water

how is sample of aqueous solution prepared for GC, GC-MS, GC-MS/MS?2

solvent extraction with immiscible organic solvent into which analyte partitions

solid phase extraction and elution with organic

how does ionisation change with purity? what doe this mean about MS signal?

what is this effect called? show graph

analytes ionise more efficiently when pure

bigger signal in MS than if it co-elutes with other species (suppressing ionisation)

• matrix effect

why is an internal standard better than an external standard for GC/LC-MS?

what is used as an internal standard? why?

due to changes in response factor of instrument

deuterated form of analyte is used as it is affected by ionisation suppression in exactly same way as the analyte

when is internal standard added? why

what is it now called?

before all work up steps

allows for losses during sample prep process

• surrogate standard

when choosing a deuterated internal standard,

why should you use more than one D? what about for molecules with Cl?

acidic or basic group considerations?

single D in place of H increases m/z by 1 so overlaps with peaks due to isotopic partners (13C)

for Cl, avoid 2xD due to isotopic partners, and for 2 Cl avoid 4xD

don’t put D on an acidic or basic group as it may be exchanged during work up and chromatography

what is multiple reaction monitoring?

like SRM, but more than one parent daughter combination

how does sample come out of chromatography column in LC, and how must it be prepared for MS

leave column at relatively high conc in liquid

• need to remove a lot of liquid to do MS

electrospray ion source for LC-MS

label

• how is solvent removed

• how are analytes ionised

• how are ions separated, how do ions change

• where do ions go after

charged droplets spray into drying chamber where they repel each other

ions shrink as solvent evaporates

how does electrospray ion source change in a glass capillary

solvent evaporates completely and leaves only gas phase ions

what are the three different modes of operation for LC-MS?

what can you use for these three?

full scan(3D output)

general detector - total ion chromatogram or base peak chromatogram

selective detector - selective ion monitoring, extracted ion chromatogram

what is base peak for positive ionisation?3

what are the sources of these ions?

M⁺ (if inherently cationic/zwitterionic)

MH⁺

MNa⁺

H⁺ from water and Na⁺ from traces in water/tubing

what is base peak for negative ionisation?3

M^- if inherently anionic/zwitterionic

(M-H⁺)^-

adducts e.g. MCl^-

mobile phase requirements for electrospray ionisation 3

need enough water to support ionisation but not too much as it is harder to evaporate

no involatile additives

MP acidified (source of H⁺ for MH⁺ ion) - e.g. with formic acid (HCOOH)

where does collision induced fragmentation often occur (ie which bond breaks)

at a single bond between carbon and electronegative element

what form must instrument sample be in when injected into instrument for reverse phase HPLC

in a solution of analyte in mobile phase (water/ co solvent)

how is aqueous solution prepared for reverse phase HPLC (2)

direct injection

or solid phase extraction and elution with polar co solvent