Mass spectrometry 1- Introduction to MS and basic concepts

Question 1

Hvad er formålet med masse spektrometri?

Answer 1

Formålet er at veje molekyler, men dette er svært fordi molekyler er små. Derfor kan molekyler ikke vejes på en vægt i laboratoriet, da de ikke er grundige nok. Her bruges istedet mass-to-charge ratio (m/z)

Question 2

Hvad er definitionen af spektrometri

Answer 2

Spektrometri er aktiviteten med at registrere og måle spektre

Question 3

Hvad er masse spektrometri?

Answer 3

Masse spektrometri er en analysemetode der kan bruges til at bestemme masse til ladning ratio (mass-to-charge ratio m/z) for et eller flere molekyler i en opløsning.

Question 4

Hvilke krav skal opfyldes under MS?

Answer 4

• Det skal være muligt at kunne danne ioner ud fra enten organiske eller uorganiske materialer. (skal kunne ioniseres)
• Det skal også være muligt at separere ionerne, så man kan finde deres mass-to-charge ratio (m/z)
• Man skal kunne måle dem (detect). Dette kan gøres ved at manipulere ionerne ved enten magneter eller elektriske felter (electric fields).
• Der skal være høj eller ultra høj vakum for at man undgår kontaminering fra omgivelser, som kan ødelægge data.

Question 5

Hvordan manipuleres ioner under MS?

Answer 5

Ved magneter eller elektriske felter, dog bruges oftere elektriske felter, da magneter ikke længere er nødvendige i moderne metoder.

Question 6

Hvorfor bruges oftere elektriske felter frem for magneter?

Answer 6

Magneter kan godt manipulere alene, men kan ikke accelerere eller fokusere ionerne alene. Derfor bruges ofte elektriske felter i kombination med magneter.

Question 7

What is the principle of MS?

Answer 7

Firstly there is a sample inlet where the sample is put into the machinery. Further there is an ion source where the sample is ionizes. Further again there is the mass analyzer. This step is where ions er separated based on their m/z. Lastly the ions are detected and shown as a spectre. All this is done in high or ultra high vacum.

Question 8

What are the overall requirements for mass spectroscopy?

Answer 8

• Fundamentals (fx ionization processes)
• Matter and mass (fx isotopic mass)
• Technical aspects
• Spectral interpretation
• Applications
  Instrumentation (fx types of mass analyzers)

Question 9

What is the definition of the mass of an element?

Answer 9

The sum of the protons (Z, atomic number) and neutrons (N)

Question 10

What is the definition of mass number (A)?

Answer 10

Z +N

Question 11

What is monoisotopic elements?

Answer 11

Elements that only consists of one actually occurring isotope.

Question 12

Hvad er en isotop?

Answer 12

Isotoper en atomer med samme grundstof (altså samme antal protoner), men med forskelligt antal neutroner, altså derfor forskellig masse.

Question 13

What is di-isotopic elements?

Answer 13

Elements that only has two naturally occurring isotopes.

Question 14

What is the formula for a di-isotopic element with two occuring isotopes with 1 u increase?

Answer 14

They are refered to as (X+1) or (M+1) elements. fx hydrogen (^1)H, (^2)H (Deterium or heavy hydrogen), carbon (^12)C, (^13)C or nitrogen (^14)N, (^15)N.

Question 15

What is the formula for a di-isotopic element with two occuring isotopes with 2 u increase?

Answer 15

They are refered to as (X+2) or (M+2) elements, e.g. chlorine (^35)Cl, (^37)Cl or bromine (^79)Br, (^81)Br

Question 16

What is the definition of relative atomic mass?

Answer 16

The weighed average of the masses of isotopes of an element. (the masses found in the periodic table) given in dalton (Da)

Question 17

What is the definition of the nominal mass?

Answer 17

Integer mass of the most abundant isotope of each element. (the isotopes with the highest %, without decimals)

Question 18

What is the definition of the monoisotopic mass?

Answer 18

Sum of masses of atoms in a molecule using the most abundant isotope of each element. (with decimals)

Question 19

What is the definition of the average mass?

Answer 19

Sum of the average masses (i.e. rel. Atomic mass) of the constitiuent elements of a molecule divided by the number of isotopes. Given in atomic mass unit (u)

Question 20

What is generally said about the mass for all different definitions of masses?

Answer 20

When the mass increases the difference between the different mass calculations increases.

Question 21

Why is the calculation of the isotopic pattern difficult for molecules bigger than 10^3 u?

Answer 21

As the variations of the relative abundances grows, it gets a lot more complicated because the peaks will blur into each other, and the most abundant mass and the average mass may shift up or down by 1 u.

Question 22

What is a good rule of thumb when interpreting isotopic distributions in mass spectra?

Answer 22

When the monoisotopic peak (containing only the lightest isotopes) is the most abundant, the isotopic distribution is easier to interpret.
As molecular mass increases, the monoisotopic peak may decrease or disappear, and the most abundant peak shifts toward the average mass, reflecting the contribution of heavier isotopes like ¹³C, ¹⁵N, etc.

Question 23

What does isotope enrichment mean?

Answer 23

• Isotope enrichment is when you add a specific isotope to a molecule, so it get more abundant to that isotope.
  e.g. ¹³C-enriched glucose is used to trace metabolism in cells. This makes it possible to track where the carbon atoms go and how the cell utilizes nutrients – a method that would not be possible without isotope enrichment.

Question 24

Name the differences of m/z and their following charge states

Answer 24

• Δm/z=1 the charge state is M^(+.) (single charged)
  Δm/z=0.5 the charge state is M^(2+) (double charged)
  Δm/z=0.33 the charge state is M^(3+.) (triple charged)
  Δm/z=0.25 thr charge state is M^(4+) (quadruple charged)

Question 25

A mass spectrometer supports the analysis of:

Answer 25

ions

Question 26

The results of a mass spectrometric analysis are given in which unit?

Answer 26

mass-to-charge ratio

Question 27

Indicate the atom below with two isotopes important for mass spectrometric analysis Carbon, oxygen, sodium, nitrogen

Answer 27

Carbon and nitrogen

Question 28

Calculate the nominal mass of methane (CH4)

Answer 28

16, Nominal mass of CH₄=12+(4×1)=16u

Question 29

What is the two major modes of ionization?

Answer 29

- Gas phase ionization by energetic electrons (M+e^.→M^(+.)+2e^−) - Protonation and deprotonation by donor/acceptor (wether the proces is protonated/deprotonated can be controlled by pH) Protonation: (M+H^+→[M+H]^+) Deprotonation: (M+OH^−→[M−H]^−+H_2 O)

Question 30

What is the difference of hard gas phase ionization and soft condensed phase ionization?

Answer 30

- Hard Ionization occurs in the gas phase and involves high energy, often resulting in fragmentation of the molecule. (often produces an radical fx M^(+.)) - Soft condensed phase ionization happens as the sample is transferred from a liquid or solid phase (condensed phase) to the gas phase using low energy, minimizing fragmentation (often deprotonized/protonized in a "box").

Question 31

Name the most used Soft condensed phase ionization methods

Answer 31

- Matrix assisted laser desorption/ionization (MALDI) Electrospray ionization (ESI)

Question 32

Name the most used hard gas phase ionization method(s).

Answer 32

Electron ionization (EI)

Question 33

What is a profile spectrum in MS?

Answer 33

It is a spectra showing the m/z over the realative intensity with noise and is showed as peaks represented as "small hills". It can show you jow accurate your analysis is.

Question 34

What is a centroid/bar graph in MS?

Answer 34

It is a spectra of the m/z over the relative intensity shown as columns. This method is more simple to read but there is a risk of loosing more information about the analysis.

Question 35

Which of the following ionic species would you expect to observe using hard ionization? Molecular ion species, protonated species, alkali adduct species

Answer 35

Molecular ion species

Question 36

Which units are commonly observed as the depedant variable (on the y-axis) in a mass spectrum? Intensity, chemical shifts, absorbance, counts, relative abundance

Answer 36

Intensity, counts and relative abundance

Question 37

The mode of ionization will affect: The m/z values observed in the mass spectrum,The stability of the molecular analyte, The degree of fragmentation, The occurence of adducts

Answer 37

The mode of ionization will affect all of them.

Question 38

What does resolution and resolving power show?

Answer 38

How well peaks are separated from each other.

Question 39

What is the formula for resolution?

Answer 39

R=(m/z)/(Δm/z)

Question 40

Which kinds of resolution definitions are there?

Answer 40

- R_(10%), describes the peak at 10% of the intensity. The formula is R=(m/z)/(Δm_(10%) ). Here Δm_(10%) is the peak width at 10% intensity. Used to define how much 2 peaks overlaps. - Full width at half maximum (FWHM), it is given as: RP=M/ΔM here the M is the maximum peak while ΔM Is the peak width at 50% intensity.

Question 41

What happens for peaks with higher m/z values(from low to high m/z) if the resolution is held constant?

Answer 41

The higher the m/z value the more isotopes overlap therefore it will be more difficult to separate and therefore read the peaks, due to overlap of the peaks.

Question 42

What will happen to the peaks if the m/z value is fixed while the resolution is increased?

Answer 42

- With increased resolution the the peak will get clearer and is easier to read.

Question 43

What does resolution affect?

Answer 43

- The ability to separate closely spaced ions. The mass accuracy, the higher resolution the better mass accuracy.

Question 44

What is the formula for determine the needed resolution?

Answer 44

Here Δm/z is the difference in m/z value for the two peaks. (the highest represented first)

Question 45

How do you calculate the accuracy of the mass (both methods)?

Answer 45

- Absolute accuracy=m/z_experimental −m/z_calculated Relative accuracy=(m/z_experimental −m/z_calculated )/(m/z_calculated ) ∗10^6 ppm

Question 46

What is the difference of Accuracy and precision?

Answer 46

- Accuracy describes how far the measured value is from the true value. - Precision expresses random error and describes how likely you are to get the same measurement upon repetition.

Question 47

How can you obtain high mass accuracy, name the methods?

Answer 47

- By calibration: -External mass calibration- done by applying reference compounds prior to the sample -Internal mass calibration- mixing the sample with a known compound

Question 48

What happens if you use to much sample in MS?

Answer 48

The composition of the isotopes can be shifted. This can give huge problems if you want to identify a compound in the sample.

Question 49

The resolution is independent of the m/z range in which you are analyzing true or false

Answer 49

true

Question 50

At a given resolution, the ability to seperate two adjecent peaks is independent of the m/z range in which you are analyzing true or false

Answer 50

false

Question 51

For two adjecent peaks (m/z=987.98 and m/z=988.03), calculate the resolution required to efficiently seperate the two peaks in a mass spectrum. Round to nearest integer number

Answer 51

19760.6

Question 52

The random error of measurements and repetability are related to: accuracy or precision

Answer 52

precision

Question 53

The devation of measurements from true values is related to: accuracy or precision

Answer 53

accuracy

Question 54

How does the calculated m/z- value from soft ionization differ from the one of hard ionization

Answer 54

During hard ionization electrons are ejected, therefore the m/z value corresponds closely to the exact mass of the uncharged molecule. While in soft ionization electrons are ether protonated or deprotonated and therefore higher or lower than the exact mass (addition/subtraction of H+)