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
What type of analyser (separator) does tandem mass spectrometry use?
quadrupole ToF analyser
- detects more than just ionic species as ions are temporally separated + can be manipulated in other ways
- e.g. in MS/MS, the ions are allowed to fragment or subjected to a second collision/ionisation event creating a new spectrum for each ion
- we can then detect which peak is for what ion
IR
Why does infrared spectroscopy work?
molecule absorbs infrared photons = change in the vibrational state of the bonds
- different bonds absorb at diff. frequencies, allowing a spectra to be obtained
units for the frequency of IR spectra vibrations?
cm⁻¹
How do we obtain an infrared spectra?
- solid sample prepared as a thin layer (film) on IR-inactive support
- pass IR radiation through sample
- waves bounced through mirror, pass through prism and detected
- IR waves absorbed by sample= not detected, therefore we detect what doesn’t make it through the detector
- things not absorbed generate a ‘dip’ - we can work out what caused this
whats detected in infrared spectra?
IR waves absorbed by sample= not detected, therefore we detect what doesn’t make it through the detector
- things not absorbed generate a ‘dip’ - we can work out what caused this
What are 2 examples of IR-inactive support?
- KBr discs
- NaCl plate (nujol gel needed for solids)
6 different ways that bonds vibrate depending on the frequency?
symm stretching antisymm stretching scissoring rocking wagging twisting
Describe the wavenumber and nature of an OH/NH bond
- ~3000-3600cm⁻¹
- broad due to H-bonding
Describe the wavenumber and nature of an C-H bond
- ~2800-3100cm⁻¹
- narrower, more defined
Describe the wavenumber and nature of an C=O bond
- ~1600cm⁻¹
- strong, sharp (almost touching baseline)
Describe the wavenumber and nature of a fingerprint region
- <1500cm⁻¹
- good for diagnosis, if we already know what compound is and can match it
Describe the wavenumber and nature of an C=C aromatic bond
- ~1800-1400cm⁻¹
- strong, sharp
3 strengths of Mass spec
- gives detailed info, esp coupled to other info
- requires VERY little material
- HRMS can determine molecular composition
3 limitations of Mass spec
- sensitive to sample preps and precise technique used
- not generally quantitative
- wont pick up impurities that ionise poorly
2 strengths of IR?
easy/cheap to run
some diagnostic capacity
Limitation of IR?
provides limited data relative to other methods
what increases ppm value?
if H is attached to carbocation
Will fragmentation of bigger peak more more likely secondary or primary structure in mass spec?
secondary: more likely to form = higher peak!
potential fragments form form breaking weakest bonds, and consider stability of the fragments!
Analysing Mixtures Using H NMR:
How to work out a mixture ratio
- find a known proton environment in each molecule
- measure the integral for an environment in each molecule
- calculate integral for ¹H in each molecule
- compare for ratio
After finding the compound ratio, what can you work out? (3)
- the molar ratio (same as compound ratio)
- molar % of one of compounds
- %wt/wt taking into account RMM
helps to ensure purity of sample
Analysing Mixtures Using H NMR is not useful for determining? (3)
levels of non-H containing species
levels of components that are not fully solubilised in NMR sample
levels of many many components with many protons (overlapping signals)
Analysing Mixtures Using H NMR: what other 4 things could you even look at?
purity of a sample?
ratio of X:Y?
ratio of drug:impurity?
ratio of metabolites?
molar % of one of compounds how would you find this (analysing mixtures H1)?
mol%= num mol of compound 1 / TOTAL num mol for BOTH
x 100
wt/wt % of one of compounds how would you find this (analysing mixtures H1)?
wt/wt%= wt of compound 1 (for given num of mol)/ TOTAL wt for BOTH (for given num mol)
x 100
Solid state: X-Ray Spectroscopy
What is a crystal?
solid material whose constituent atoms/molecules/ions arranged in ordered pattern extended in all 3 spatial dimensions
difference between crystal shapes? (plates, cubes, needles, etc.)
microscopic patterned order of atoms/molecules/ions results in a specific macroscopic order; these give plates, cubes, needles, etc.
Is kinetic solubility influenced by the formulation or is it an innate property of the molecule?
formulation:
- solution dosing (because drug needs to be in solution to be absorbed)/salt forms/amorphous forms all increase rate/extent of dissolution
- alternatively enteric coating etc. can delay/control dissolution
Is thermodynamic solubility influenced by the formulation or is it an innate property of the molecule?
innate property of molecule:
- varies at varying pH, where ionisation improves aqueous solubility
- still has kinetic component as rate of precipitation can be low for poorly soluble molecules too
name of processes of formulated drug -> solution drug -> solid drug
dissolution
or precipitation from 3->2
Why does it matter as to whether we’re dealing with the non-crystalline/amorphous form of the compound or the polymorph state of the compound?
- energy associated w/ breaking the crystal lattice = key component of thermodynamic solubility (∆G)
- non-crystalline (amorphous) material has a lower lattice enthalpy, and therefore is likely to have a high solubility
- compounds with multiple crystal states (polymorphs) can have diff physical properties, e.g. solubilities and therefore diff pharmacokinetic properties
solubility of non-crystalline (amorphous) material?
non-crystalline (amorphous) material has a lower lattice enthalpy, and therefore is likely to have a high solubility
What electromagnetic interaction that X-ray diffraction and spectroscopy involve?
X-rays scattered by atoms - tells us what crystalline form sample is
What within a unit cell of crystal diffract x-rays and why? (2) What is this technique called?
single crystal X-ray:
- the ordered arrangement of heavy atoms creates ‘planes’
- these planes diffract x-rays due tot he electron density of the atoms
What is the the x-ray diffraction pattern from a single crystal composite of and how is this useful? (single crystal X-ray)
- the diffraction of all the planes within the crystal
- get mostly destructive interference, however in certain places get constructive interference which makes a clear pattern
- pattern used to understand the electron density pattern of atoms in the crystal; 3D arrangement of atoms in unit cell and hence structure of molecule
- this technique can be applied all the way up to proteins
What information does single crystal X-ray give us?
- structure of molecule due to arrangement of electrons in compound
- determination of molecular interactions in solid state which can give the polymorph structure
- e.g. a and b have H bonding but in different parts of the molecule
What is inconvenient about single crystal X-ray?
time consuming process, requires preparation of large ‘perfect’ crystals
- generally crystals used in drug formulation are smaller
What can we use instead of single X-ray patterns?
a powder - X-ray powder diffraction patterns
Why do X-ray powder diffraction patterns work?
- although there is a less distinct diffraction pattern due to the random arrangement of particles, there’s still defined planes within the sample due to the presence of crystalline solid
- the planes can scatter the light at defined angles from the beam, resulting in distinct rings around the sample (as opposed to spots w single X-ray)
- observing different angles of diffraction shows us that the energy of the diffracted X-rays change, giving an XRPD (fingerprint) pattern
What do X-ray powder diffraction patterns look like?
i.e. what is plotted?
- angle of diffraction plotted against intensity
- specific to each polymorph
- each line = intensity of line of pattern
- can help us match up polymorphs to check whether they’re the same or different
spiky
What do X-ray powder diffraction patterns look like for amorphous material and why?
more of a downward curve not spikes
-no defined planes to give rise to the peaks normally seen, resulting in undefined diffraction patterns
Solid State-Techniques - Part 2
What are two alternative methods to differentiate between polymorphs or amorphous material instead of X-ray spectroscopy?
- Differential Scanning Calorimetry (DSC)
- Thermogravimetric analysis (TGA)
What is differential scanning calorimetry (DSC) and why do we use it instead of melting point observations?
- crystalline nature of a solid has significant impact on its melting point (due to difference in lattice energy needed to break apart a crystalline form)
- using conventional melting point observations is not accurate enough for characterising polymorphs
DSC:
Any e_ or e_ event in the sample will change the energy required to maintain the temperature gradient
any endothermic or exothermic event in the sample will change the energy requirement to maintain the temperature gradient
What does a DSC graph look like?
power (change in energy consumption) plotted against temperature
flat with 1 peak
what does DSC measure?
difference in amount of energy (heat) req to increase temp of sample and reference at a constant rate
Explain how an endothermic event would affect a DSC graph.
- initially, straight line as same amount of power needed to keep increase the energy at a constant rate
- this constant is changed when an endothermic event occurs as it raises the temperature
- in order to increase the temperature beyond that constant rate, we need to put in more energy/power
- therefore some of energy goes into increasing the temp whereas some goes to the endothermic event
What could enthalpy of fusion (melting area, triangle Hf) look like on a DSC graph?
- more energy/power needed to heat up sample, break up solid bonds and melt sample
What other examples of transitions (apart from enthalpy of fusion) could DSC graphs show?
ENDOTHERMIC:peak/bump up:
- loss of water from hydrate
- glass transition
- loss of absorbed solvent
- Melting area=enthalpy of fusion
EXOTHERMIC:peak/bump down:
-isothermal
-crystallisation/form change
triangle Hc
difference in exo and endothermic
endo: take in energy, positive power
exo: give out energy, neg
whats TGA (thermogravimetric analysis)? how does it differ from DSC?
extension of DSC, similar but also includes accurate assessment of mass of sample over range of temp in DSC
what can help differentiate between diff events in DSC?
TGA!
allows separation of mass neutral events (change in form/melting) from other events - desolvation and or decomposition
TGA can offer what type of results?
quantitative measure of amount of solvent released durign a desolvation event
water etc
why can TGA also cause and observe combustino of organic compounds?
as its able to be used at high temps
measuring Co2 release –> mass lost
how does amss change when solvent change from solid -> liquid (TGA)
no change in mass, straight line on temp/ power (% mass change) graph.
only dips when dolvent loss = reduction 25% in mass of sample
key strengths of:
single crystal X ray spectroscopy?
allows atomic, molec and 3D crystalline struc of a compound to be elucidated.
(e- density)
definitive
key strengths of:
XRPD?
allows assessment of crystallinity of solid material
useful for assessment of polymorphs (not amorphous)
key strengths of:
DSC?
gives accurate and quant analysis of energy changes in samples as function of heating
useful to determine certain elements: MP, desolvation etc
key strengths of:
TGA?
useful to quantify and identify mass changes, esp as result of gain/loss of solvent
key limitations of:
single crystal X ray spec?
need rel large amount material of good quality and large crystals
(not wanted rlly and need right conditions to grow)
key limitations of:
XRPD?
doesnt provide interpretable data beyond matching of diffraction patterns
not ideal for amorphous
key limitations of:
DSC?
material may undergo changes on heating (changes in polymorph) = complex and misleading data.
multiple types of events detected on single scale, hard to interpret
key limitations of:
TGA?
expensive and rel low throughput
older machines, not accurate inn quantifictaion
UV-Vis Spectroscopy:
What part of the EM spectrum does UV-Vis spectroscopy involve?
UV/Visible light region (2-900nm) - molecular electronic transitions
Radiation in the UV-Vis region of the EM spectrum has the correct energy to do what and how?
- it can promote/ excite electrons in lower orbital into higher energy
- the photon of (UV) light is absorbed, transferring energy to an electron in molecule which becomes excited to a higher energy orbital
What does the wavelength of light absorbed by an electron directly correspond to?
the energetic gap between orbitals
(however spectrum produced is a continuous profile as energy levels in molecules are complicated by conformational/vibrational effects)
on UV spec graph what mols are in
- lowest energy (bottom)
- middle
- highesr energy (top)
- normal “bonding” pairs of e-
- lp: “non bonding”
- normally empty orbs “anti bonding”
How is a UV-Vis spectrum obtained?
a sample in solution is used and compared to a sample of solvent alone
What does a UV-Vis spectrum indicate and why is it difficult to analyse?
- frequency of light absorbed, which is needed to promote an electron between 2 electronic states
- the peak can be quite broad, making it difficult to assign parts of the spectra to the specific particles of the molecule
What law explains why UV-Vis absorption is so important?
the Beer-Lambert law
A=e c L A = absorbance a = molar absorptivity L = length of light path c = concentration
How does the Beer-Lambert law explain why UV-Vis absorption is important?
- states that difference between light absorbed + transmitted is proportional to absorbance of the compound
- the second part of the equation also states that absorbance is equal to εcl, where ε is specific to a compound
How does the Beer-Lambert law help us to determine the concentration?
- know: ε is constant for a certain compound
- the path length (l) is constant as it’s measured by the spectrophotometer
- we measure I and I₀
- therefore the only thing that will change is the concentration (c) which is directly linked to absorbance
so:
c dir.prop to A
What is the first issue with using UV absorption, relating to the nature of absorption of UV light?
- wavelength of light absorbed links directly to energetic gap between orbitals e.g. σ/σ* transitions require high freq short wavelength waves and therefore absorb poorly
- simple molecules with not a lot of conjugation (alkanes, isolated alkenes, carbonyls) absorb weakly at short wavelengths
- instead, those going from π to π* (antiboding) are absorbed really strongly, so conjugation in a molecule produces strong spectra
What is a chromophore?
the part of a molecule that absorbs light
Molecules with what absorb more strongly and at higher wavelengths? (2)
- extended π systems (e.g. conjugated aromatics)
- conjugated aromatics have alternating single/multiple bonds in some parts of the structure
=-=-=-
not =–=—
What type of molecules is UV useful for evaluating, given that σ/σ transitions absorb weakly but π/π absorb strongly?
aromatic-type compounds or compound with conjugated systems
= luckily most drugs
What is the most common wavelength used to evaluate the UV absorption of drug molecules, and why?
- 254nM
- this is the frequency for a benzene ring, which is contained in many drug molecules
Effect of conjugation between amino acid Trp (most conjugated), Tyr (less conjugated) and Phe (least conjugated)
- Trp has strongest absorbance corresponding to conjugation within indole
- Tyr has lone pairs from OH conjugated into aromatic system, giving strong absorbance too
- Phe only has benzene ring, but the line would be completely flat without the ring
Why are tomatoes red?
- contain mg quantities of lycopene, which is a long conjugated system giving strong absorbance
- it absorbs at 440, 470 and 502 which is the blue region, so reflects colours in the red region
wherever peak = absorbed and NOT COLOUR REFLECTED/SEEN
What is pulse oximetry?
- the measure of %Hb loaded with O₂ to give an estimate of %arterial O₂
- a pair of LEDs facing a photodiode through a transculent (fingertip, earlobe) body part
- 1 red LED (660nm) and one IR LED (940nm)
- as oxygenated and de-oxygenated blood absorb differently in the EMC spectrum, the 2 absorbance values can be compared
- this comparison is proportional to the relative concentration of the 2 species
- ratio is converted to %O₂ by a Beer-Lambert law processor
What is the second issue relating to the spectra of UV-Vis spectroscopy?
how is this problem overcome?
- compounds with a good response can have overlapping spectra
= hard to evaluate mixtures - cant tell what peak came from what compound - for this reason, it’s combined with another technique that can separate/identify components in a mixture
- therefore UV-Vis spectroscopy is very powerful when combined with chromatography
How is combining UV-Vis spectroscopy and chromatography useful?
- separate compounds using chromatography, then use UV-Vis to analyse the spectra
- as conc = proportional to the absorbance, UV becomes a powerful quantitative technique to measure the concentration of components in a mixture
- can be done by separating the mixtures, measuring the absorbance of one UV-Vis wavelength (as this will be specific to the compound), then comparing this info to standard (calibration) curves
Fluorescence:
What electronic transition can molecules undergo instead of looking at the UV-vis absorbance spectrum? What are molecules able to do this said to have?
- a non-radiative relaxation of electrons, resulting in the emission of a light photon with lower energy than that absorbed
- this light emission has lower energy/wavelength than that absorbed and is most noticeable when the light emitted is in the visible range
- molecules that can do this have a fluorophore
What are the two spectra fluorescent molecules have?
one for light absorption and one for light emission
Why do we set up the sensor to measure the emission spectrum perpendicular to the light we’re putting in (excitation source)? 90 deg
- to reduce the signal-to-noise ratio
- this ensures the light emitted that we detect comes from the sample and not the original light source
higher wavelentgh = lower energy = fluorescence
in fluoresce, whats the difference in wavelength/ peaks of absorption and emission called?
and what is it characteristic of?
stokes shift
characteristic of the flurophore
Systems with larger delocalised systems absorb/emit what type of energy?
fluoresc
- lower energy
- longer wavelengths
What is the absorbance spectrum of resorufin and what does it show? Why does we get a bright pink solution when we put resorufin in solution?
- the light absorbed promotes electrons to a higher orbital around the bluey-green region
- therefore the reflected colours red and small amounts of purple to give pink
What is the emission spectrum of resorufin?
- some red, completely covered orange, some yellow and bit green
- therefore the light emitted is bright orange
peak shifted to right slightly
How can we use the absorbance and emission spectra of resorufin?
- we take a green laser pointer (region of absorbance) and shine it at resorufin
- this causes electrons to be excited, then they fall back down and emit the wavelength of orange light
How does shining a green laser pointer at resorufin help us?
show whether cells are alive and turning over or not
- we can see the production of resorufin from resazurin which we feed our cell, as resazurin is reduced to resofurin by aerobic respiration of metabolically active cells - confirms cells are viable
- then we shine the green light and see if it emits an orange colour
- (can also be oxidised to dihydroresorufin but this is reversible, and can be oxidised again easily)
look at pic!!
How is fluorescence useful for quantifying concentration?
in some fluorescent drugs, the emission is more specific than absorption e.g. ciprofloxacin levels in urine
resazurin = reduced to resorufin by aerobic resp of metabolically active cells- can be used as indicator of what?
cell visibility.
also sense mitochon activity
detect NADH/NADPH,
assay L-Glutamate among others
How is fluorescence useful for monitoring interactions?
e.g. GFP and variants
- compound with a fluorophore can be linked to another system e.g. protein
- this can also indicate if a drug hit is interacting with the protein in question
How is fluorescence useful for FACS machines?
FACS = Fluorescence Activated Cell Sorting Machines
allow us to count number of cells in solution that we’re putting in solution, hence cell sorting
What is chromatography?
technique for the separation of a mixture by passing it through a medium in which components move at different rates
- the mixture will separate based on how well it interacts w the solid phase vs the mobile phase
What is termed the mobile phase?
The solution that the mixture is in or the separation medium where the solution is passed?
the solution that the mixture is in
What is termed the solid phase?
The solution that the mixture is in or the separation medium where the solution is passed?
the separation medium where the solution is passed
Chromatography can be used for what 2 purposes?
analysis or separation (purification)
What are the 5 main methods of chromatography?
- paper chromatography
- thin layer chromatography (TLC)
- column chromatography
- HPLC (high performance/pressure liquid chromatography)
- LCMS (liquid chromatography mass spec)
In chromatography of a mixture, why does separation occur?
components have different affinities for the mobile and solid phases
In the chromatography of a mixture, which component will move more slowly?
that with higher affinity for the solid phase (red)
In the chromatography of a mixture, which component will move more rapidly?
that with lower affinity for the solid phase (blue) - interacts with bar less than it interacts with solution
think red carpet, unknown celebs
What 2 techniques visualise the components as they lie on the stationary phase?
paper chromatography and TLC
How does paper chromatography work? (describe the solid and mobile phase)
- paper is made of cellulose= lots of free OH groups making the solid phase very polar and able to interact (Van der Waals, H bonds) w polar compounds
- therefore more polar compounds compounds (w more heteroatoms/HBDs/HBAs) will move more slowly
- the mobile phase can be any solvent but is typically a H2O/EtOH mix
paper chromatography: what dye will stay close to bottom?
those with more polar compounds as interacts with polar paper more :)
How does thin layer chromatography (TLC) work? (describe the solid and mobile phase)
- solid phase is silica coated plate (glass/aluminium plate w silica sprayed on) = polar
- mobile phase is organic solvent
- therefore the more polar the compound (more heteroatoms/HBDs/HBAs), the more it will interact w solid phase and move up less
similar to paper
How is thin layer chromatography carried out?
- run solvent up to certain height, pull out, then draw where solvent got up to
- put under UV light if UV active or react w compounds like permanganate to see it change colour
What is an Rf value and what form of chromatography does it correspond to?
- retention factor value: distance travelled by compound divided by distance travelled by eluant
- TLC
2 dp.
always less than 1!
What does the Rf value show us about the compound? and hence is linked to…
affinity of the compound to the silica plate, which can give an indication about the functionality in a compound
- hence it’s linked to the polarity of a compound
What are Rf values specific to?
- the solvent system the plate is run in
- changing the solvent system changes the Rf value
If we change the solvent system ratio in TLC so that it becomes more polar, what happens to the components?
material will move further up as plate has increased affinity to solvent
difference between TLC and column chromatography?
- identical concept
- instead of visualising ‘snapshot’ of compounds on plate, keep letting them elute off the end
- solvent is collected until it drops out the end in fractions
- most non-polar component (at the top of the TLC plate) will elute off first (as solvent is organic/non-polar)
difference in TLC and column chormat in terms of use?
- TLC is more for analysis, and column chromatography is more for separation (we can obtain the pure compound)
What is normal phase chromatography? Name 3 examples
- techniques which use a polar stationary phase and non-/less polar mobile phase
- TLC, column and paper chromatography
What are the 3 limitations of normal phase chromatography?
- polar phase can degrade w repeated used (e.g. OH groups on silica are reactive so can become capped and results become inconsistent)
- polar stationary phase can become contaminated w polar impurities, so unideal for high throughput analysis
- requires use of flammable/toxic organic solvents e.g. hexane
What can be achieved by reversed phase chromatography?
- we reverse the polarity of the phases: polar mobile phase and non-polar stationary phase
- by this we use a system w a more inert stationary phase, making weak interactions w mixture components and a more acceptable mobile phase that’s e.g. water-based
In reversed phase chromatography, what is the stationary phase and what is the mobile phase?
- stationary phase: still silica based but hydroxyl groups chemically modified w long chain alkanes (most commonly C18) making it very non-polar
- mobile phase: water mixed w less polar solvent (acetonitrile + 1% trifluoroacetic acid to ensure acids protonated and in neutral form) so non-polar compounds now
Which compound is retained more in reversed phase chromatography: the non-polar or polar?
the non-polar as the stationary phase is now the very non-polar/lipophilic silica hydroxyl groups capped with C18 chains
Which compound travels further in reversed phase chromatography: the non-polar or polar?
the polar as the mobile phase now contains water and the stationary phase is extremely lipophilic
Which compound is retained more in reversed phase chromatography: the non-polar or polar?
What techniques most commonly use reversed phase chromatography?
- HPLC
- LCMS
- sometimes TLC
How does HPLC (high performance liquid chromatography) work?
- similar to reversed phase TLC/column chromatography
- separation achieved by diff affinities for mobile/solid phases (which is reversed in this case)
- solvent continually pumped through column based on X18 silica
- sample added into stream as conc. band of sample in solution
- substances separated on column and detected at end of stream
What do the physical elements of HPLC/LCMS experiments look like?
- solvent sits at top of machine
- sample loaded into small vials and used w autosampler
- solvent goes through switching valve, then column, then detected by detector
- fractions collected w bottle on side and linked to computer
What does an example of a reversed phase chromatogram look like? Describe the axes. What is the retention time? What does it depend on? In the image, which interacted better with the column?
intensity/time
2 peaks (2 components)
- retention time depends on interactions w stationary phase, hence structure
- pink has a lower time so interacted less w column and came out a lot quicker
chromatograohy:
whats retention time tr and what compounds have lower tr?
retention time (tr): how long it takes from injection of mixture onto column until compound reaches detector
lower time if interacted less w column and came out a lot quicker
What value is retention time (tr) similar to
The Rf value in TLC
What is the designated tm value?
i.e. what does it take into account?
this value takes into account the minimum time for any unretained material (usually mobile phase) travels through column
What is tr’?
- adjusted retention time
- it’s for a solute and it’s the additional time for a solute to travel the length of the column (compared to designated tm)
tr’ = tr - tm
What formula connects tr’, tr and tm? What does it take into account?
tr’ = tr = tm
- i.e. taken away time for NO compound to get through machine from how long it took for our compound to get through machine
- takes into account length of column
Why is HPLC/LCMS diagnostic?
- the same compound will have the same retention time
- because every time it is run it will interact w the solid phase vs the solvent to the same extent assuming the conditions are identical
- can therefore identify the compound if you have a standard run of the isolated material
Chromatography paired with what technique is useful for quantifying the concentration of an unknown?
- UV-Vis, due to the Beer-Lambert law, which states absorbance is proportional to concentration
- the area under the curves (AUCs) in HPLC/LCMS is compared
How can UV-Vis and HPLC be combined to be quantitative?
- UV-Vis spectroscopy is difficult to tell signals apart due to overlap
- if signals are separated, then use absorbance of 1 wavelength of UV-vis light, then use AUC of each peak and that will proportional to concentration of component
- HPLC quantitative when used in combo with the extinction coefficient (which is different for each compound
- also quantitative by comparing result w AUC of a peak from a sample of known concentration, and comparing to standard curve
Example of overlap in UV-Vis spectra and why the extinction coefficient is needed (amino acids)
- may get 280nvm from Tyr or trp
- from UV-Vis, tyr would give less of a good absorption
- this overlaps with Trp as it may look like a lot is being absorbed by Tyr, but it’s due to trp
- therefore E is needed as it’s different per compound
Why are there limits of quantification with UV-Vis analysis and standard curves? What is the region between these called?
calibration/standard curve generated to ensure absorption relates accurately to concentration:
- at low concs, signal-to-noise ratio becomes significant, creating lower limit of quantification
- at high concs detector will saturate, creating upper limit
- region between these limits if the linear range, where absorption or AUC can be reliably used to get concentration
What does the upper and lower limit of quantification look like on a standard curve?
- bottom 2 values very close together
- top value very far off
- results in low R-squared value
For reliable results on a HPLC chromatogram, we need to be able to tell the difference between the AUC for all the peaks. This means they need to be..
… well resolved with no overlap: the mathematical formula states the difference in retention times need to be bigger than the width of the peaks that could overlap (which is half the total width of the peaks)
How does the resolution of peaks produced by columns w larger silica particles and smaller surface area compare to those produced by columns w smaller silica particles and a larger surface area?
Larger:
- larger spaces between particles
- compounds spend a lot less time in the column
- broader peak
Smaller:
- smaller spaces and larger SA:V so can pack more into the same space
- compound forced to interact more w silica particles rather than dodging through
- sharper peak
therefore although the AUC is the same, the smaller particles give a better resolution
price of improving resolution by decreasing silica particle size?
- smaller gaps between particles means higher pressure required to push solvent through column
- this gave HPLC its name high-pressure liquid chromatography
What is meant by isocratic?
the column in HPLC is eluted with a single eluent
What is the issue with using isocratic solvent systems?
- using an isocratic gradient is necessary to find an eluent of correct polarity to separate all components of a mixture
- however, it would give broader, asymmetrical peaks with low resolution because the solvent only has one polarity
What can we use instead of an isocratic solvent system?
- a gradient solvent system, using a polarity gradient
- this means starting with an aqueous eluent and slowly increasing the organic component until the final stage of column is in organic solvent
- blue is isocratic, red is gradient solvent system
How does using a polarity gradient reduce overlap?
- you increase the amount that the compound interacts w the solvent as it goes along the column
- this stops peaks trailing (blue) and sharpens them up (red)
- it also improves versatility as it can be used on more mixtures that previously overlapped with isocratic conditions
Visualisation of isocratic vs gradient solvent system
- isocratic would be problematic if the column was really short and stopped in the middle
- gradient has good separation towards end due to acceleration of compounds (travelled far)
What is meant by LCMS and use?
liquid chromatography mass spectrometry:
- helps use get a better feel for the identity of compounds being separated by pairing HPLC/UV to a MS spectrometer
- allows for simultaneous generation of a mass-trace, providing info on the molecular composition of peaks absorbed in UV, and presence of UV-inactive impurities
When is LCMS only quantitative?
in relation to standards by measuring the UV-Vis, as mass spec is not normally quantitative either
What can HPLC be paired with to ensure peaks are clean compounds?
MS/MS - we figure out what the compound peak corresponds to, then use MS/MS to check the peaks don’t overlap
What can you tell about a HPLC/UV-vis spectrum done on a mixture? (example w standard response spectrum next to it)
- how many components it may contain e.g. in example, contains at least 3 components due to 3 peaks (some may not be separated or UV-active)
- it potentially contains same component as that on standard response on right (but can’t be sure due to the possibility that another compound has same polarity and hence retention time)
- that with the longest retention time is most non-polar (due to interacting more with non-polar stationary phase - reversed phase)
- mixture has lower drug concentration than 5uM compared to standard, as the AUC is lower
What can’t you tell about a HPLC/UV-vis spectrum done on a mixture?
cannot be certain which compound has highest concentration due to peak height, as each compound has different extinction coefficient
bond angles of sp2 and sp3 C
sp2 C = 120°
sp3 C = 109.5°
key strengths of:
UV-Vis spec?
provides quantitative, linear evaluation of conc
C dir.prop to A
can make comparisons therefore
key strengths of:
HPLC
- used in urine/blood samples
- allows assessment of conc in mixtures (vs standards) using UV-Vis
- high throughput/low cost and low sample requirements. dont need pure sample
key strengths of:
LCMS
- allows assessment of conc in mixtures (vs standards) using UV-Vis/ specialised mass spec
- provides some info on composition of peaks
- may observe UV-inactive components.
mass out, no UV trace
key limitations of:
UV-Vis spec?
- poor separation of mixtures
- not useful for analytes w weak/no chromophores (only for pure sample)
key limitations of:
HPLC?
- gives little data on nature of any analytes observes
- with UV detection any UV-inactive components will not be observes
relying on UV-Vis
key limitations of:
LCMS?
- only quantitative in relation to standards (UV AND specialised mass spec)
- struc info limited in same way as MS
dont know comp of parent ion/cmopound
how can you tell difference of what is causing broad peak in IR spec? at 3200-3600: NH or OH?
you cant!
but question may specify compund only has C,H,O in it etc..
what is fluorescence?
when some molecules absorb energy i.e. electronic transitions.
Most suitable methods to know if given drug has been metabolised normally in particular patient/s
HPLC (UV/MS)
And
HPLC (UV)
Which analytical method would indicate if the API (active pharmaceutical ingredient) is in the correct physical form?
X-Ray Powder Diffraction
In DSC graph, what is likely source of first peak?
Desolvation of water
which analytical method most suitable for info on content of blood sample?
NMR LCMS IR XRPD DSC
LCMS?
on NMR, what mols will be on LEFT side of ppm graph? (3)
deshielded
sp2 more electroneg
OH (as more e-neg than NH)
