5CCC2010 Advanced Chemistry for Life Sciences (22~23 SEM1 000001) CHEAT SHEET
53 views 0 purchase
Course
5CCC2010 Advanced Chemistry (5CCC2010)
Institution
Kings College London (KCL)
Choosing advanced chemistry was one of the worst descions of my life. As much as I like chemistry, chemistry does not like me. If you feel the same- this document is for you!
This double sided A4 "cheat" sheet contains ALL and I REALLY MEAN ALL of the content from the whole of the semester. It i...
Spectroscopy o Useful for fingerprinting of complex biological environments Shifts in Absorbances – Solvatochroism o Secondary structure gives characteristic absorptions
- Determines the structure of a molecule Surface Enhanced Raman Scattering (SERS) - Solvatochromic behaviour – absorbance is dependent on the polarity of the solvent it Ramachandran Plot
- Can improve Raman signal using nanoparticles that have surface plasmon resonances is in
Importantly, allows for the rapid detection of molecules and allows us to quickly access - Proteins are a mixture of secondary structure therefore need to quantify CD spectrum
changes in structures. o E.g. Au/Ag nanoparticles - Change in absorbance arise from how a solvent stabilises the ground (or excited) state - Mean residue ellipticity (MRE) can be used to plot data on absolute scale
What is spectroscopy? o Attach analytes to the surface or have nanoparticles in close proximity of the molecule - Can be deconvoluted to give approximate contributions of secondary structure
Interaction of electromagnetic radiation with matter Nanoparticles for SERS biosensing o Changes energy gap between them
→
Absorption = matter receiving energy from radiation - Fewer stretches visible in Raman than FTIR o
Emission = matter releasing energy as radiation o Easier to pick out signals in complex environments
[ Θ ] 222=−9500 ,Φ α ,222=0.20
- The more polar the solvent the more stable the zwitterionic ground state
Excitation from ground state to excited state is quantised. - Nanoparticles can be used for SERS in biological environments
o Fluids/cells
→ →
= have discrete bands of energy
- Targeted delivery allow detection of biomarkers without needing to purify
the larger the HOMO-LUMO gap absorbance undergoes blueshift
c
- Can be used for cancer
UV/Vis Spectroscopy (towards UV) o
λ= Molecular Orbitals
- Molecules are collections of atoms connected by shared electrons – bonds
Change the molecule, Change the absorbance
[ Θ ] 208=−8962 ,Φ α ,208=0.17
v
- A molecules absorbance can often be changed by performing simple chemical
- Bonds form by combination of atomic orbitals to give molecular orbitals reactions
- Atomic orbitals in phase = Constructive overlap - Small changes in conjugation can have a big effect on the absorbance
1
o Bonding molecular orbitals o These empirical relationships can use MRE at 222nm and 208nm to estimate the
- pH indicator e.g. Phenol Red
~v= - Atomic orbitals out of phase = Destructive overlap
o Anti-bonding molecular orbitals
o
o
pH>8.2 = red
pH<6.8 = yellow
α-helical content
Thermal stability of Proteins
λ
- Anti-bonding molecular orbitals are formed simultaneously with the bonding - Proteins are minimally stable structures
- para-Nitrophenol
molecular orbitals o ~10kJmol-1
o goes from colourless to yellow under basic conditions, forming p-nitrophenolate
- HOMO = Highest Occupied Molecular Orbital - The folded form exists in equilibrium with the denatured form
o p-nitrophenolate can be incorporated into model compounds, so that after a
hc - LUMO = Lowest Unoccupied Molecular Orbital o Under physiological conditions, the equilibrium lies in favour of the folded state
E=hv= =hc ~v
bond cleavage, p-nitrophenolate is released
- Molecular orbitals only form when the atomic orbitals are close in energy (e.g. N2) o Easily shifted by temperature, chemical denaturants solvents
Allows you to monitor the reaction and measure rate
- Non-bonding orbitals (or lone pairs) occur when atomic orbitals are far apart in energy Lipase - Temperature dependent (CD) spectra gives information on the thermal stability of
λ or are of incompatible symmetry (e.g. HF)
o p and s orbitals cannot combine therefore cannot form a bond
- Molecular orbitals apply across the whole molecule – the bigger the molecule, the
- Enzyme catalyses the hydrolysis od fatty acid esters
- Important for many industrial biotechnology applications
what is being measured
o With the correct experiment, Kc can be calculated which can lead to the free
energy
Wavelength ∝ Energy o Can be found in “bio” laundry detergents
more complex the molecule (e.g. Benzene) - Equilibrium requires 2 distinct states: beginning and end
IR Spectroscopy Measuring Reaction kinetics
UV/Vis o To determine the equilibrium, first need to pick a point that changes with the
- IR part of the spectrum is at higher wavelength therefore lower energy than UV/Vis - If reagent is colourimetric – releases a coloured product – kinetics can be measured
- Measures the absorbance of Ultraviolet and Visible radiation temperature (or other reactive conditions)
o Vibrational Excitation using UV/Vis spectroscopy
- The molecular orbitals is what absorbs the light and gives its colour o Wavelength chosen should ideally be representative of the molecule
λ max
- IR gives the range where bonds stretch and bend
o Most likely transition is HOMO-LUMO Transition o Plot the signal intensity at that wavelength against temperature (or other
- Usually plotted as transmission against wavelength (cm-1) - Example, Lipase, = 410nm
Typical energies = 125-650kJmol -1 conditions)
- Plots are arranged in order of wavelength
Typical wavelength = 200-800nm o Calculate fraction denatured (or generally, the fraction of product/end point in
o Higher wavenumbers to the left o Measure absorbance at 420nm with time
- Allowed electronic transitions occur from occupied orbitals to unoccupied orbitals the equilibrium
ε
- Motions available for bonds, all of which will be present in an IR spectrum
(bondto antibond) o Convert to concentration using - Using (temperature dependent) spectroscopy, it is possible to determine
- Symmetric stretching, Asymmetric stretching, Twisting, Wagging, Scissoring, Rocking
Vibrational Structure thermodynamic parameters without need for calorimetry
- When bonds vibrate, they behave like a spring
[ θ ] 208 + 4,000
- Absorptions only occur within strict selection rules between ground and excited state o Direct measurement of reaction kinetics
o Can be described by Hooke’s Law
o However, when molecules absorb electromagnetic radiation, the excited state o ∆A = ∆C = rate
Therefore, stretching frequency depends on strength of bond and mass of atoms either
end of the bond
o
causes bonds to be longer, caused by electrons in the antibonding orbitals Heme Proteins
Therefore, each electron transition is associated with change in vibrational and - Proteins that contain a heme prosthetic functional group as its functional group ϕ α , 208=
rotational states -> allows multiple transitions
Electronic transitions faster than vibrational transitions
o Heme = iron containing Protoporphyrin IX
- Key blood proteins – Myoglobin and Hemoglobin
29,000
Etotal =Ee Supramolecular Chemistry and Molecular Machines
o Iron porphyrin connected directly via histidine residues
−¿ o Active site allow for reversible binding of O2 Organic Chemistry
vib
+E ¿ Oxidises Fe 2+ to Fe3+, causes a structural shift - Organic chemistry is the chemistry of organic compounds
As stretching motions are higher energy than bending motions - The number of compounds containing carbons is greater than the number of
Franck-Condon Principle
¿
π ¿π
Chromophores - Porphyrins (when bound to proteins) have a very strong absorption due to compounds of all the rest of the elements in the periodic table combined
- Parts of a molecule that contain bonds that will absorb in the UV/Vis regions - One of the reasons for this complex chemistry is that carbon, with its 4 valence
- All molecules will have electronic absorptions electrons – can form 4 covalent bonds
Bonds as Harmonic Oscillators o Strongest istransitions
Soret band at around 400nm followed by the Q bands at higher
¿ - Carbon is also able to bond to itself to form long chains, branched chains and cyclic
σ →σ
- There are selection rules for transitions wavelengths
o Most transitions are too high in energy to be observed on structures
o Selection rule #1 ∆v±1
¿
π ¿π
o Allows large biomolecules such as proteins, lipids, carbohydrates and nucleic
o Selection rule #2 (for interactions w/IR) Must be a ∆ dipole moment o The transitions are very sensitive to changes in structure
typical spectrometer (<200nm) acids
( 12 )hv π
- It covers many broad areas of research including:
o Double and triple bond ( ) and Lone pairs (n) are observable Fluorescence Spectroscopy
E= v +
o Theoretical organic chemistry – understanding structures in terms of atoms and
Photoluminescence
electrons that bond them together
Absorption (A) - Chemiluminescence – light emitted from the decay of the excited state (e.g. Luminol)
o Synthesis – how to design and make new molecules
- The change in intensity (I) of light as it passes through a sample o If this reaction is biological, its bioluminescence (e.g. Luciferin in luciferase found
o Structural determination – how to find out the structure of new compounds,
I0
in fireflies)
even if they are only available in small amounts
- Bonds don’t obey Hooke’s law exactly o T1 state to S0 state
o Reaction mechanism – how the molecules react with each other (individual steps)
A=log
- Force required to compress a bond is larger than the force required to stretch it - Similar set up to UV/Vis spectroscopy
o and how to predict their reactions
- Bonds, if stretched enough will eventually break (dissociated) - Measured orthogonally (90˚) to the path of the excitation beam
I
o Biological activity – find out what nature does and how the structures of
- Can describe the energy using a Morse Curve - Need to pick excitation wavelength to get best emission spectrum
- Fluorescence detection tends to be more sensitive than absorption biologically active molecules are related to what they do
o At low energies, vibrations are essentially harmonic Hydrocarbons
Fluorophores
I0
But become more anharmonic with increasing energy - Simplest organic compounds contain just H and C – known as hydrocarbons
Energy levels get closer together = the initial light intensity - Fluorophores can be excited by chemical reactions rather than light
- Typically large organic molecules with highly conjugated bond system - Broadly divided into 3 categories:
Vibrations of Polyatomic Molecules o Aliphatic – not cyclic or aromatic; only straight, or branched chains
- Can be functionalised or incorporated into other molecules
I
- Large molecules have a more complicated stretching and bending modes – Normal Saturated – Alkanes (CnH2n+2)
Modes = the light intensity at the end o Fluorescent part is called the fluorophore
- Large range of molecules = large range of excitation + emission Unsaturated – Alkenes (CnH2n), Alkynes (CnH2n-2)
o Synchronous motion of atoms that may be excited without exciting any other o Alicyclic – cyclic compound that are not aromatic; rings with no delocalised π-
modes - Attenuation of light intensity will depend on how strongly the sample absorbs, how - Very sensitive to environment – can be used as a molecular probes
much of it is present (concentration), and how far light has to travel through the - Fluorescein (Fluorescein isothiocyanate system
o Non-linear = 3N-6 Saturated – Cyclic alkanes (CnH2n)
o Linear = 3N-5 sample (pathlength) o Basic fluorophores but robust molecules with a high fluorescence intensity
The Beer-Lambert Law o Used as a dye that can be tagged onto other molecules Unsaturated – Cyclic alkenes (CnH2n-2)
o 2 or more modes may be degenerate (have the same energy) o Aromatic – compounds with benzene rings or related systems; has a ring with a
Measuring IR spectra Absorbance (at a given wavelength, unitless) can be quantified with this equation: o Used in medical imaging
delocalised π-system
A=εcl
- Measured using a spectrometer - BODIPY
Functional groups
o Vibrational absorbances are not measured at every wavelength o Very sensitive to chemical modification
- Group of atoms in organic molecules that are responsible for characteristic chemical
- Fourier Transform o Very versatile fluorophore
- Build a calibration curve of known concentrations if extinction coefficient is not known - Nile Red reactions of those molecules
o An interferogram of IR radiation is collected
ε
Orbital Hybridisation
o Converted to a spectrum of frequencies using a Fourier Transformation o Has different forms but Nile red most common
o Gradient of A vs. C gives - Carbon can form up to 4 covalent bonds
FTIR – Fourier Transform InfraRed o Very sensitive to chemical environments – particularly polarity
o However, the modern electronic structure of carbon show there are only 2
Preparing samples Absorption in biomolecules – Proteins o Used as a molecular prob for polarity
unpaired electrons that could be contributed to covalent bonds
- To minimise background, samples are prepared with as high a concentration as - Main a.a. to look at is Tyrosine (Tyr/Y) and Tryptophan (Trp/W) - Molecular Rotors – Fluorescent Viscosity Probes - Formation of bonds releases energy so the system is more stable
possible o Are distinguishable o Greater viscosity, greater fluorescence o Forming 4 bonds rather than 2 releases more energy = more stability
- KBr pellet o Tyrosine (Tyr/Y) - GFP – Green Fluorescent Protein o Only a small energy gap between 2s and 2p orbitals
λ max
o Made by compressed sample with KBr under high pressure o Isolated from jellyfish
o The energy required to promote an electron from 2s to 2p to give 4 unpaired
- Nujol mull =274nm o Natural fluorophore
electrons is compensated by extra energy released from forming 4 bonds
o Very concentrated emulsion of sample and Nujol (mineral oil) o Can be genetically modified to be different colours - The electrons then rearrange themselves again in a process called hybridisation
ε 274
- More common – Attenuated Total Reflectance (so ATR-FTIR) o Can be incorporated into other living system o Organised into 4 identical hybrid orbitals, called sp3 (from 1xs and 3xp orbitals)
o Samples loaded onto a diamond plate and measured directly = 1400M cm -1 -1
o Made up from 3 amino acids (Ser, Tyr, Gly) to make one chemical structure o Arranged themselves in space so that they are as far apart as possible – angle of
IR Sampling Methods o Excitation of the deprotonated form gives greater fluorescence intensity 109.5˚ (Tetrahedral)
ε 280
- Transmission Fluorescent Microscopy - Alkenes (unsaturated carbons)
o Bulk measurement = 1280M -1cm-1 - Multiple fluorophores can be used to probe and image systems o Only joined to 3 other atoms than 4
o KBr discs o Chemical modify fluorophores to tag different parts of the cells o Only 3 orbitals hybridised, rather than all 4
o Nujol mulls o Tryptophan (Trp/W) - Hoechst – Binds to DNA in cell nucleus o Arranged as far as possible – 120˚ to each other in a plan (Trigonal planar)
o Thin films
- Specular reflection λ max = 280nm o λ ex = 350nm
Remaining p orbital is at a 90˚ to them
o Surface measurement - Hybridised sp3 and sp2 (or sp) orbitals form
σ bonds
o Thin films on reflective surfaces
- Diffuse reflection
o Surface measurement
ε 280
= 5690M -1cm-1 o λ em = 460nm - p orbitals can overlap to form π orbitals
- Aromatic compounds
- PKH67 – Binds to cell membrane o Electrons are delocalised, which stabilises the compound
ε =nW ( 5690 ) +nY ( 1280 )
o Powdered samples
λ ex
- Attenuated Total Reflection o Bond length the same (140pm instead of 147 for C-C or 135pm for C=C)
o Surface measurement o = 490nm All the C-C bonds are midway between a single and double bond
ε
o No sample prep Nonpolar and polar covalent bonds
- The surface measurements may not always be great as the surface molecules may be - If number of Y and W is known in a protein, you can calculate
λ em
- Nonpolar covalent bond = bonded atoms are the same, or have similar
different to the internal molecules o = 502nm electronegativities – electron density is evenly shared between atoms
o You can get this from the primary sequence - Polar covalent bond = bonded atoms have significantly different electronegativities –
FTIR spectra
- Typically measured as transmittance(%T) against wavenumber o Measure absorbance at 280nm - Rhodamine Phalloidin – Binds to actin filaments (cytoskeleton) electron density is shifted towards one atom
λ ex
o Inverted axis (y-axis) o Calculate concentration using Beer-Lambert equation o The difference in electron density leads to partial charges either end of the bond
o If Mr of protein is known, concentration can be calculated in mgmL-1 −¿¿
- Can convert transmittance to absorbance: o = 540nm
+¿∧δ ¿
A=2−lo g 10 ( %T )
Absorption in biomolecules – Nucleotides – represented by
δ
o - ε 260 [ Adenosine monophosphate (AMP/A)] = 15,400 M-1cm-1 o λ em = 565nm o Difference in charge is known as a dipole
Reaction classification
FTIR Imaging
Provides more detail to images – chemical composition with spatial resolution
- Cancer Fingerprinting
- ε 260 [ Guanosine monophosphate (GMP/G)] = 11,500 M-1cm-1
Fluorescence of Proteins
- Tryptophan – weakly fluorescent
o Intensity increased when its in a hydrophobic environment
- Polymerisation – joining together lots of simple molecules to form a giant molecule
- Substitution – when one species is replaced by another
- Addition – when one species is added to another
o Nucleic acid – Phosphate group
ε 260 o Used as a measure of protein folding - Condensation – when two species joined together with the loss of water
o Proteins – Amide group
λ ex
- [ Cytodine monophosphate (CMP/C)] = 7,400 M -1cm-1 - Hydrolysis – splitting a molecule into 2 molecule by adding water
o Lipids – C-H group o = 380nm - Oxidation – when an atom loses electrons
Proteins
ε 260
- Reduction – when an atom gains electrons
- Diverse set of natural polymers – distinct structures
λ em
- [ Thymodine monophosphate (TMP/T)] = 8,700 M -1cm-1 - Redox – any reactions where electrons are transferred between 2 species
- Increasingly important for molecular sciences and biotechnology o = 300-350nm Electrophiles and Nucleophiles
o Viruses – vaccines Nucleic Acid Concentration - Nucleophile (nucleus/positive lover) – a reactant that provides a pair of electrons to
o Antibodies – immunotherapy - Concentrations of DNA and RNA are easier to determine than protein form a new covalent bond
o Temperature dependent tryptophan fluorescence
o Enzymes – biocatalysts o All nucleobases absorb o Has a -ve charge, partial -ve charge or a π-bond
Used to probe changes in tertiary structure
- Structure very important – dictates function o Have slightly more bases than average proteins have amino acids - Electrophile (electron/negative lover) – a reactant that accepts a pair of electrons to
Tertiary structure denatures before secondary structure
o Difficult to assess quickly - Generic Extinction coefficients: forma a new covalent bond
Can be monitored by CD spectroscopy
Secondary structures
ε 260 - Protein aggregation (forming fibrils) is the cause of neurodegenerative diseases o Has a +ve charge, partial +ve charge or an empty valence orbital
- Proteins are polymers of L-amino acids connected by peptide bonds o [dsDNA] = 0.020mlug -1cm-1 o Aggregation creates new hydrophobic domains Mechanisms
- Typically no structure - Arrows show the movement of electrons as bonds between atoms are broken and
o Fluorophores can be trapped in aggregated fibrils
o Described as being “random coils” or “unordered”
ε 260 Increase in fluorescence formed
- Amino acids can hydrogen bond to each other o [ssDNA] = 0.027 mlug-1cm-1 o Single headed arrow = movement of one electron
CD Spectroscopy
Hydrogen bonding patterns o Double headed arrow = movement of a pair of electron
Circular Polarised Light
- α-helix
ε 260
- Light can be polarised so that the plane of the polarised light (perpendicular to the - This is a formalism – electrons (more precisely, electron density) do not move around
o H bonds between amino acids 4 residues apart on same peptide strand o [ssRNA] = 0.025 mlug-1cm-1 so neatly and discretely in reality
direction of wave) rotates at a steady rate
- Β-sheet o Can be left or right handed - Rules:
o H bond between adjacent peptide strands Shifts in Absorbances – Conjugation 1. Arrow must start from electrons and point to an atom or bond
- Chiral molecules will absorb left and right handed light differently
The peptide bond - Molecules with multiple chromophores have greater extinction coefficient o Absorption will depend on orientation of the chiral bind 2. Electrons and bonds form bonds, not charges
- All proteins have an abundance of peptide bonds
π
o Repeated dihedreal angles will give stringer absorption o However, common convention in organic chemistry is to use a shorthand
o Very visible stretching in IR - When chromophores are conjugated (usually -bonds) notation of a -ve charge to represent a lone pair in mechanisms – for charged
o The difference in absorption = Circular Dichroism
- Amide I and II are the strongest of the vibrational modes atoms either show all lone pairs or none at all
o Characteristic peaks for each give secondary structure
Amide I Amide II
o
o
Energy between HOMO and LUMO decreases
Absorbance undergoes a redshift Δ A= A L −A R Supramolecular Chemistry
- “Chemistry beyond the molecule”
λ max ε
- The most important feature of supramolecular systems is that the components are
α-helix
β-sheets
1648-1657cm-1
1623-1641cm-1
1540-1550cm-1
1525-1535cm-1
o As number of double bonds increase, values of and increase
Θ=32.98 Δ A held together by reversible, intermolecular forces, not by covalent bond
- Important concept in supramolecular chemistry is complementarity e.g. base pairing
- Retinal, bound to protein rhodopsin, main receptor for vision – absorbs light CD of Proteins in DNA double helix
Unordered/Random 1642-1648cm-1 1535-1540cm-1 o Only aldehyde form is active however, greatest conjugation (more redshifted so - Proteins are polymers of L-a.a. connected by peptide binds o Combines 2 antiparallel strands held together by complementary H-bonds
→
o Typically, no structure, “random coil”/”unordered” between pairs of bases
Raman Spectroscopy UV Visible) o Can H-bond to each other o H-bond donors and acceptors are arranged that adenine forms 2xH-bonds with
→
- Also measures vibrational states thymine, whilst guanine forms 3xH-bonds with cytosine
o Many birds and insects (particularly bees) can see in UV - R group dictate allowed dihedreal angles very specific secondary structure - A receptor (the host) is complementary to the substrate (the guest)
o Uses a form of Rayleigh scattering
Change in polarity of the binding pocket of rhodopsin so is blueshifted – o Its size, shape, and position of binding sites are ideal for substrate recognition
- Provides complementary knowledge to IR
o Show vibrations that do not break a molecules symmetry
- There a fewer transitions
Glutamic acid
→ tyrosine/phenylalanine
- Peptide bonds is chromophore – chiral and planar
o Repeated dihedral angles give string absorptions
o “lock and key” principle
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller Boce. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $26.50. You're not tied to anything after your purchase.
