Analytical Flashcards
define:
a) precision
b) accuracy
a) agreement between set of results (question of error)
b) how close is result to ‘true’ answer
What are 2 ways to improve the precision of data and why?
- run analysis multiple times
- use SOPs (standard operating procedures
both reduce the potential for human error
What does accuracy rely on the generation of?
standards - repeated on the day of analysis or at beginning and end of each batch of samples to ensure performance of method is maintained
What must machines be to maintain accuracy? (c….)
calibrated
What is the robustness?
the method’s reliability in changing conditions e.g. can it be done in colder temperatures, etc.
What is spectroscopy?
study of interaction of electromagnetic radiation with matter
What part of the EM spectrum concerns NMR?
radiowaves
What 4 observations can be made from ¹H NMR?
1) number of signals
2) chemical shift (position of signal)
3) integration
4) peak splitting (coupling)
What information can we get from observing the number of signals?
the number of ¹H environments
What information can we get from observing the chemical shift (position of signal)?
the type of ¹H environment
What information can we get from observing the integration?
the number of ¹H of each type
What information can we get from observing the peak splitting (coupling)?
the number of adjacent ¹H
Why do spectra contain more than 1 peak for all hydrogens?
- magnitude of ΔE = dependent on size of the magnetic field experienced by the nucleus
- the multiple peaks tell us not all protons experience the same magnetic field
- due to the e- surrounding the ¹H nuclei; generate an electric current that shields the nucleus lowering the magnetic field strength experienced, changing ΔE
How does electronegativity affect the nature of a ¹H NMR peak?
- Hs attached to electron-withdrawing groups/atoms have a lower share of electrons
= nuclei experience higher magnetic field due to less shielding from less e- density
- this increases the ppm value, moving the peak downfield
How does electronegativity affect the nature of a ¹H NMR peak?
study of interaction of electromagnetic radiation with matter
What property of fundamental particles/intrinsic form of angular momentum does NMR utilise?
spin
property of spin is fundamentally linked to…?
magnetism
some nuclei also possess imbalanced spin states -> magnetic properties essential for NMR!
What property of fundamental particles utilised by NMR is seen in electrons?
spin - electrons occupy orbitals in pairs with opposing spin states
What are 6 examples of atoms with an overall spin, and why do they have it?
¹H (proton NMR), ¹³C (13C NMR), ¹⁹F, ¹⁴N, ¹⁰B, ²H (deuterium)
all possess an odd number of protons and neutrons resulting in imbalanced spin
Why don’t ⁴He, ¹²C, and ¹⁶O possess an overall spin?
they don’t have an odd number of protons and neutrons
need odd number for spin!
NMR relies on the relationship between s_ and m_
spin and magnetism
How are the spin angles of nuclei in atoms normally oriented?
What happens when we apply a magnetic field?
Are the molecules free to move?
- randomly orientated
- become aligned with/ against magnetic field
- molecules can still move freely but w nuclei in defined orientation
What is the energy state of nuclei that align AGAINST the magnetic field?
the higher energy state known as beta (β)
What is the energy state of nuclei that align WITH the magnetic field?
the lower energy state known as alpha (α)
What does the energy gap between the alpha and beta states correspond to?
the electromagnetic waves in the radio frequency (60-1000 MHz (ΔE = hν)
the energy released when beta state nuclei relax/alpha state nuclei become excited
What does the energy gap between the alpha and beta states mean for nuclei (transition)?
nuclei in alpha state can absorb radiowaves at certain freq and become excited to beta state
excited beta state nuclei can relax to the alpha state and release this same frequency of energy - this is ΔE and what’s is looked at to observe the 1H in the molecule
What do we do to ΔE after it’s released from beta state nuclei relaxing?
- Its emitted in form of a radiowave + detected using a radio receiver
- signal complicated due to other spins also relaxing back down
- signal therefore undergoes extensive mathematical manipulation (Fourier Transformation) to give spectrum displayed as intensity against δ chemical shift (ppm).
How do we run an NMR experiment in practice? 6 steps
- Sample dissolved in deuterated solvent, placed in NMR tube
- Placed in NMR spectrophotometer with strong magnet
- Sample spun to even out imperfections in sample
- Sample irradiated w/ pulse of radiowave-frequency radiation
- Once pulse finished, nuclei relax to low energy state and emit radiowave radiation- detected
- Results undergo mathematical transformation and display results as intensity against δ chemical shift (ppm).
what happens to nuclei during step 4 of NMR experiment?
Sample irradiated w/ pulse of radiowave-frequency radiation
spins will align with/against energy field
What 2 things does the magnitude of ΔE (frequency of radiowaves absorbed/emitted) depend on?
1) the size of the magnetic field experienced by the nucleus - higher field strength increases energy difference between states and hence produces better signals
2) the nucleus - energy differences and spin state complexity (e.g. different amount of energy need to excite different spin states to higher energy state) means we can only look at 1 type of nucleus at a time
Why do we need to standardise NMR spectra?
different NMR machines have different magnetic fields (field strengths), which can affect the magnitude of ΔE
How do we standardise NMR spectra machines across different machines?
- they are NOT displayed as frequency (Hz) on the x-axis
- instead displayed as a ppm shift (parts per million, written as δ) relative to a reference compound, TetraMethyl Silane (TMS, SiMe₄), which is given δ = 0 ppm.
Do we measure the sample while it’s solid or in solution?
in solution
Why do we need to use special solvents to to dissolve our sample in for NMR? What are these special solvents?
- sample needs to be in solution
- most solvents contain H atoms, and these signals would be seen as the solvent is in excess
- instead we use deuterated solvents
What are deuterated solvents and why can we use them instead of normal H-containing solvents?
- all hydrogens have been
replaced by deuterium (²H - like hydrogen but with an additional neutron - deuterium nucleus itself is spin active and so magnetic, but absorbs in a very different frequency to ¹H so the solvent is no longer seen in the spectrum
What are 3 examples of deuterated solvents and what do each of them dissolve?
- CDCl₃
- D₂O
- DMSO
All have different properties:
- CDCl₃ can dissolve organic substances
- D₂O can dissolve water-soluble substances
- DMSO good option when other solvents cannot dissolve the substance
What is the problem with deuterated solvents?
exchangeable protons:
- protons in sample can exchange w deuterium in deuterated solvent
= these protons disappear from NMR spectrum/ don’t integrate properly (as a mix of visible ¹H and invisible ²H)
What types of protons are likely to become exchangeable when being dissolved in a deuterated solvent?
- any protons attached to a heteroatom (esp acidic/ basic sites), aka most likely to dissociate
- deuterated solvents that can also dissociate are not exempt from this e.g. D₂O, d₄-MeOD
What medical technique is closely related to NMR?
MRI:
- machine is giant magnet
- protons will excite
- depending on where they are in body tissue will determine the frequency of radiowave
- this allows us to tell the difference between tissues without invasive procedures
As you go downfield a spectrum/increase the ppm value, how does the delta charge and shielding on that proton nuclei change?
it becomes more deshielded and delta positive, higher ppm
why do electron withdrawing groups= higher ppm?
low e- density around proton = deshielded= higher mag field = higher ppm (more left)
What are the general ¹H NMR trends in protons (0.0-10.5ppm)?
i.e. order: sp2,3 Cs: alkenes, alkanes…
highest ppm:
protons on…
- sp2 C next to e- group (O, aldehydes)
- sp2 C: benz, arom hydrocarbs
- sp2 C: alkenes
- sp3 C next to e- grou (O)
- sp2 C: CH, CH2, 3
lowest ppm
What protons are found within the 0-3.0ppm range? why?
protons on sp³ carbons such as CH, CH₂, CH₃, carbon chains (no electronegative atoms so can experience a lot of shielding)
Why doesn’t paracetamol give out 9 peaks despite having 9 protons in its structure?
hydrogens in equivalent environments give the same signal as they’re shielded/ deshielded by the same amount and give out the same ∆E when they relax
(symmetry)
What is the integral? and what does it relate to?
area under the curve, relating directly to the number of H atoms in the same in that environment
What is splitting?
Hs in peaks interact with neighbours, resulting in peaks with multiple peaks
How is splitting/coupling caused?
most commonly seen between non-equivalent Hs separated by 3 bonds
(through bonds, not space)
H-C-C-H
What is the peak splitting and ratio of 1 adjacent hydrogen?
a doublet, with a ratio 1:1
What is the peak splitting and ratio of 2 adjacent hydrogens?
a triplet, ratio 1:2:1
H environment is adjacent to 2 protons, causes 3 energy levels
What is the peak splitting of 3 adjacent hydrogens?
a quartet, ratio 1:3:3:1
How can peak splitting be predicted?
using the n+1 rule:
- no. of peaks = number of adjacent hydrogens +1
=Splitting is not seen through what kinds of atoms?
heteroatoms, as it’s not always consistent through these
How many environments in cyclopropanol and why?
- 4
- the 3D shape of the molecule means blue protons are on same 3D side of OH, whereas red protons are on same side as pink proton
how does a peak split due to nearby protons look?
symmetrical (Pascal triangle)
if not, it may be 2 peaks overlapping!
Is ¹³C NMR more sensitive than ¹H NMR? Why or why not?
¹³C: in much lower abundance than ¹H (only 1.1% of carbon atoms in a sample will be visible, making ¹³C NMR less sensitive
= rel low throughput and expensive as need bigger machine/ more sample and time
What are the 2 observations you should make from ¹³C NMR?
- the number of signals
- the position of the signal (chemical shift)
What information can you get from the number of in C13 NMR
the number of ¹³C environments
What information can you get from the position of a signal (chemical shift)?
¹³C NMR
the type of ¹³C environment
What types of carbon signals are found in the 0-50ppm range?
C13 NMR ORDER?
lowest ppm
sp³ carbons not next to an electronegative atom e.g. CH, CH₂, CH₃, carbon chains
sp³ carbons next to an electronegative atom e.g. CH₂OH and sp alkyne carbons
sp² carbons not next to an electronegative atom e.g. alkenes
sp² carbons next o an electronegative atom or aromatic e.g. ketones = 180-200ppm, aromatic ¹³C = 140-160ppm
highest ppm
How does the nature of the peak change with the electron density of a carbon?
the more electron deficient a carbon is, the more downfield (higher ppm value) it will be
(sp3 C not next to electroneg atom e.g. CH2, CH3 in C chains)
Does the peak size correlate to the number of carbons in the environment?
- no
- ¹³C nuclei relax at very different rates depending on the environment
What are the key strengths of ¹H NMR? (3)
- provides quantitative measurement of H nuclei in solution
- very info-rich and interpretable
- predominantly used in characterisation of molecules
What are the key weaknesses of ¹H NMR?
- not useful for impurities not containing H
- only measures material in solution
- requires mg quantities of solvent-free material
- expensive and low throughput (takes 5 mins to run a sample that gives us a small amount of data)
What are the key strength of ¹³C NMR?
useful for characterisation of complex chemical structures
What are the key weaknesses of ¹³C NMR? (2)
- not generally quantitative (cannot tell how many Cs there are)
- expensive and relatively low throughput (takes 20 mins to run a sample for a small amount of data)
What distinguishes carbon environments?
- the sequence of bonds the carbon atom has to other atoms
- if two carbon atoms have the same bond sequence they will have the same environment
What do mass spectrometers do?
generate gas phase ions from a sample, then measure the mass-to-charge ratio of these ions m/z (z value is normally 1)
e 4 main steps of mass spectrometry?
1) sample processing
2) ionisation (fragmentation)
3) acceleration + separation of ions
4) detection
What occurs in the 1. sample processing step of mass spectrometry?
- sample needs to be in solution
- chromatography performed to check for purity - HPLC (GC)
What occurs in the 2. ionisation (fragmentation) step of mass spectrometry?
- sample needs to be in gas phase in order to ionise
- can fragment
- can be done via ESI, EI, CI or MALDI
What occurs in the 3. acceleration and separation of ions step of mass spectrometry? (3)
- ions are accelerated towards detection plate
- figured out how to separate ions
- can be done via sector (deflection), waiting for how long it takes to get to detector (time of flight), or quadrupole
What occurs in the 4. detection step of mass spectrometry? (3)
- ions flown through sheet
- once they hit the sheet, = charge generated
- hence ions detected through induction of charge/current
Is mass spectrometry a form of spectroscopy?
no, as it doesn’t involve any interaction of electromagnetic radiation with matter
What is the molecular ion (M⁺)?
the ion comprising of the most abundant isotopes of elements in the molecule
What is EI?
electron ionisation - an ionisation method:
- vaporised sample exposed beam of electrons
- causes electrons to be either removed (giving M⁺∙ - unpaired electron) or added (giving M⁻∙)
Why is electron ionisation a hard ionisation method? (3)
- it’s harsh on the chemical= formation of radical ions
- tend to have low stability
- therefore they can fall apart quite easily leading to a high degree of fragmentation
What is CI?
hows it done?
chemical ionisation - an ionisation method:
- sample vaporised then exposed to beam of chemical ions e.g. CH₃⁺, CH₄⁺ formed by electron ionisation (in case of methane)
- can result in 4 types of ionisation events: protonation, abstraction, adduct formation, and charge (electron exchange)
- all these events result in an ion being produced from the original sample
4 types of ionisation events possible from ionisation method CI: what occurs in each event between M (sample) and methane ions (CH₃⁺)??
protonation,
abstraction,
adduct formation,
charge (electron exchange)
protonation,
M + CH₃⁺ → MH⁺ + CH₄
abstraction,
MH + CH₃⁺ → M⁺ + CH₄
adduct formation,
M + CH₃ → [MCH₃]⁺
charge (electron exchange)
M + CH₄⁺ → M⁺ + CH₄
What is ESI?
and hows it done (3)?
electrospray ionisation - an ionisation technique:
- sample solution exposed to high voltage
- leads to formation of aerosol consisting of small and highly ionic liquid droplets
- these droplets expel ions often MH⁺, MNa⁺
Why is ESI considered a relatively soft ionisation technique?
- less fragmentation due to more stable ions
- more likely to see initial mass of sample at end
Why does fragmentation occur in mass spec?
- initial ionic species formed in spectrometer may be unstable
- leads to degradation into smaller fragments
What does the stability of an ion relate to? (2)
- the energy of the ion
- the structure of the ion
resonance
How does the energy of an ion affect its stability and therefore fragmentation?
higher energy radicals: more likely to fragment than low energy protonated species
How does the structure of an ion affect its stability and therefore fragmentation?
structures are more likely to fragment next to polarised bonds:
- C-N bond broken
- carboxylic C-C bond broken
How do the results of EI and ESI compare?
EI is hard, ESI is soft so more likely to produce more stable fragments (adducts, protonated species)
Why is ionisation not completely reproducible?
i.e. whats it also affected by?
it can be affected be salts, solvents or other impurities in sample,
e.g. high energy radicals could be stabilised by impurities in the sample, helping them survive longer
What are the 3 analyser types used to separate the mass ions formed to allow the detector to find the m/z ratio?
- sector (deflection)
- time of flight (Tof)
- Quadrupole (AKA Quadrupole Tof)
what is sector (deflection) based on and how does it work? (4)
- deflection of ionic particles by magnetic/static electric field
- smaller more highly charged particles deflected to greater extent than larger/less charged particles
= varying the current: see different particle sizes - this degree of deflection is directly related to m/z
How do Time of Flight (ToF) analysers work?
- similar principle to sector (deflection)
- ions accelerated using electrostatic field of known strength (instead of variation like in sector)
- instead of degree of deflection, rate of acceleration controlled by m/z where small highly charged particles accelerate faster (e.g. empty trolley accelerates faster than full trolley)
- m/z therefore determined through time taken to reach detector
How do Quadrupole analyser types work?
and hows m/z determined?
- ions passed through oscillating electrostatic field generated by charged rods
- these rods oscillate the electrostatic field by changing the path of ions
- m/z determined by trajectory (i.e. the exact oscillating field applied to get a certain mass to go from entrance to exit - resonant frequency) of the ion
- as majority of ions are deflected, this allows for a narrow m/z range to be detected
in quadrople ToF analyser type in mass spec, whats the role of
a) analyser
b) detector?
a) separates ions
b) detects ions
How does a mass spectrometer detect the ions?
through induction of charge/current
whys mass spec not a form of spectroscopy?
doesnt involve study of interaction of electromegnetic radiation w matter
Why is the molecular ion peak not always the biggest?
may be due sample fragmenting due to e.g. the ionisation technique
What do the other peaks apart from the molecular ion peak correspond to?
the fragments
What are isotope patterns?
why does it happen?
- spectrums generating peaks due to isotopes
- due to mass spectrometers measuring abundance of ions, therefore mixture of isotopes within atoms of sample =detected by the MS
- e.g. With Cl where ³⁵Cl is 75% and ³⁷Cl is 25%, may get 2 peaks where one is 3x bigger than the other
Example spectra of bromobenzene (isotope patterns)
- 2 isotopes in approx 50:50 ratio
- we can use their mass to calculate which isotope the peaks correspond to
Example spectra of dibromobenzene (isotope patterns)
why is mass 236?
- 236 is due to the average from the periodic table
- 234 is only from ⁷⁹Br, 238 is only from ⁸¹Br, whereas 236 is from an average of both (2 possible ways to get this mass) creating a ratio of 1:2:1
What is the molecular ion peak always accompanied by?
isotope peak
What equation describes the ratio of [M]⁺ to [M+1]⁺ and why?
[M]⁺ to [M+1]⁺ = 100:(1.1n)
where n = number of C atoms:
- 1.1% chance that a carbon is ¹³C and not a ¹²C
- therefore in 10 carbon system there’s 11% chance of there being one ¹³C
- on spectra ratio is 52:4, leading to 100:7.69 - n - 7 (don’t always work out this cleanly)
What else complicates molecular weight apart from isotopes?
Energy:
- huge amt required to hold together protons in nucleus = strong nuclear force
- energy derived from P+N in accordance w/ E = mc²
- equation shows energy related to mass; have to use some mass to hold the nucleus together
= nuclei of atoms weigh less than the sum of masses of protons + neutrons they contain
What isotope is the standard for all atomic masses?
¹²C
What do exact mass values take into account?
the energy used to hold the nucleus together
What is HRMS and its use?
High Resolution Mass Spectrometry:
-to determine mass of an ion to >4 decimal places, allowing ion to be linked to a single molecular formula
Example of how exact mass helps us
- if we only knew that the mass was 94, we could see that it would be any of the 3 shown
- from the decimal places we can at least distinguish the 2 isomers from the one containing F
what features (3) of mass spec can distinguish isotopes?
HRMS (more dp)
isotopes
fragments
What does tandem mass spectrometry (MS/MS) help us with?
- can fragment a fragment more
- helps to determine if fragmentation relates to parent compound or impurity
