20/21 - Spectroscopy Flashcards
Some uses of SPECTROSCOPY:
Mixture of compounds -> Chromatography -> single compound
Single Compound -> KNOWLEDGE OF STRUCTURE
Bio System + Compound –> Compound Localization / MoA / Protein-protein Interactions
What is SPECTROSCOPY?
Study of matter through its interaction w/ diff compounds of the EM spectrum,
interaction of energy with matter
understand how LIGHT interacts with MATTER
how to use it to learn about variety of properties in a molecule
4 First (smallest) Wavelength Regions
& what they measure
Gamma rays - n/a
X-Rays - core/inner level electrons
UV & Visible** = **valence / outer electrons
3 LAST (largest) Wavelength Regions
& what they measure
IR** **- molecular VIBRATIONS
Microwave - molecular ROTATIONS
Radio - NUCLEAR SPIN
What does a spectrum measure?
how the sample MODIFIES THE LIGHT interacting with it
- Light source (EM wave) -> Sample
- Change in Light
-
Change in Sample
- =photochemistry
- leads to DEGRADATION, typically irreversible
- =photochemistry
Typical process of “small molecule” structure ELUCIDATION
Compound ->
IR / UV-Vis / MS ->
1D NMR / 2DNMR ->
IDENTIFIED STRUCTURE
X-Ray Cytallography SKIPS all those steps
X-Ray Crystallography
Energy Interaction / Central Concept
DIFFRACTION = bending of light around an object
-
x-ray beam -> crystal of a SINGLE PURE crystal compound
-
bend/defract/scatter light in 3D space, 360 rotation
- onto a detector, which is then computed
-
bend/defract/scatter light in 3D space, 360 rotation
X-Ray Crystallography
Used for / Works on?
ribosome / how proteins fit together / antibiotic mechanism
has to be a PURE / SINGLE crystal
(has a HUGE number of PRECISELY ordered, IDENTICAL molecules)
Performed on both:
SMALL or LARGE
molecules 100-2k Da or >2k Da
X-Ray Crystallography
strengths & weaknesses
- Strength:
- Possible to determine entire structure** including **relative or absolute stereochemistry of small molecules
Good if you can get it to crystallize in the first place
- Weakness:
- Crystallization process is laborious; certain classes of molecules are difficult/nearly impossible to crystallize
•Crystallization is difficult to make
IR (infrared) Spectroscopy
Energy interaction / Central Concept
IR spectroscopy measure
MOLECULAR VIBRATIONS
- X = Wave number (cm-1)
- Y = % Transmittance, similar to absorbance but FLIPPED
- Specific functional groups act in a
-
VERY SPECIFIC & CONSISTANT WAY
- made a reference sheet for comparison
-
VERY SPECIFIC & CONSISTANT WAY
IR (infrared) Spectroscopy
Used for / Works For?
presence of FUNCTIONAL GROUPS
Bond Distance
Natural product elucidation, but NMR/MASS spectroscopy is better
need to compare to a reference / cheat sheet
IR (infrared) Spectroscopy
Strength / Weakness
-
Strength:
-
Indicates potential presence of FUNCTIONAL GROUPS
- including bond distance
-
Indicates potential presence of FUNCTIONAL GROUPS
-
Weakness:
-
does NOT indicate CONNECTIVITY of functional groups
-
ex. we don’t know how 8 carbons + 10 hydrogens are bound,
- dont know the actual molecule STRUCTUR
-
ex. we don’t know how 8 carbons + 10 hydrogens are bound,
-
does NOT indicate CONNECTIVITY of functional groups
Atomic Absorption (aa) Spectroscopy
Energy Interaction / Central Concept
EMISSION SPECTRA of each element produces a unique fingerprint
Flame Test - similar to FP (Flame Photometry)
Flame converts metal ions -> atoms
measures Light Intensity
AA Spectroscopy
Works on / Common uses
Determination of METALS (Hg / Pb / etc)
from LIQUID** **(urine / plasma / serum)
AA Spectroscopy
Strengths / Weaknesses
-
Strength:
- __used for Metals in Liquids/Solution
-
Weakness:
-
Low throughput, requires manual operation
- _DESTRUCTIVE_
-
Low throughput, requires manual operation
UV-Vis Spectroscopy
Energy Interaction / Central Concept
- Spec of UV & Vis regions measures valence / outer electrons
- Molecules absorb EM radiation
- as atoms pass from ground state -> Excited State
- Molecules absorb EM radiation
- More effectively a molecule ABSORBS LIGHT (given wavelength,
- –> GREATER the extent of light ABSORBANCE
- views CHROMOPHORE
- functional group that absorbs UV radiation
Chromophore
Functional group that absorbs UV Radiation
views CONJUGATION
organic molecules between 190-800 nm
detected by UV / PDA Detectors
Name the Process of rapidly identifying compounds that are
KNOWN CHEMICAL SCAFFOLDS
DEREPLICATION
used in UV/PDA
UV / PDA Detectors
PhotoDiode Array
both view Light Absorption** of **Chromaphores
Photodiode Array (PDA) Detectors = pretty much the same as UV detection but with ALL WAVELENGTHS
IF DETECTABLE BY UV IT IS ALSO DETECTABLE BY PDA,
but not the other way around
UV / PDA Spectroscopy
S / W
-
Strengths:
- UV fingerprint may indicate the CLASS of a molecul
-
Weakness:
-
does NOT distinguish between ISOMERS within a molecular class
- if the structural change does not involve the CHROMOPHORE
- Need an EXTENSIVE UV fingerprint
- some classes do not absorb in UV regionn
-
does NOT distinguish between ISOMERS within a molecular class
Common Chromophores
Blue box = solvent interference
chromophore wavelength OVERLAPS
w/ wavelength of solvent
Chromophres with more complex conjugation
Complex conjugation = more DISTINCT UV Fingerprint
UV / PDA Spectroscopy
Works on / Common uses
UV fingerprint is helpful for INDENTIFYING compound class if:
MOST of the structure contributes to the UV Chromophore
but we can link a chromophore to a reactive functional group
to make an UV-Active material
ELSD
Evaporative Light Scattering Dector
Concept / Detects for?
Replacement or used in TANDEM w/ UV/PDA Detector
does not rely on spectroscopic qualities of molecule
only detects a PHYSICAL PRESENCE
of a compound, either SENSES or NOT,
needs physical blockage to be detected
ELSD
Evaporative Light Scattering Dector
Steps / Method
DESTRUCTIVE DETECTION METHOD
detects for PHYSICAL PRESENCE
Solvent + Anyltes -> METAL NEBULIZER
Evaporated / carrier gas transports MIST through LASER
Gas condenses on tube after being removed from anylites
ELSD Detection
Used For?
detects Physical Presence
useful for detecting molecules that do
NOT have any spectroscopic qualities
is destructive though
Fluorescense vs Absorbance
You can’t have _fluorescence_ without absorbance
-
Absorbance
- FIRST step in fluorescence.
- electronic transition that promotes electron from ground state (LIGHT ENERGY)
- FIRST step in fluorescence.
-
Fluorescence
- the light emitted by an atom or molecule
- after absorbance of EM energy
- the light emitted by an atom or molecule
Fluoresence
-
Light Emitted by an atom or a molecule after
- the absorbtion of EM energy
- Need to Filter OUT excited light
- remaining light is Fluorescent light
- enables visualization of molecules / proteins
- remaining light is Fluorescent light
Fluorophores
-
Molecules or Functional groups that have the
- Capacity to exhibit flourescence
- Structural characteristic of useful fluorophores:
- Extended Conjugated Pi bonds
- the MORE conj bonds
- & lower the excited energy requirement
- LONGER the wavelength (redder) the exciting light can be
- & lower the excited energy requirement
- Ex. GFP, green fluorescent protein
GFP Mechanism
Green fluoresent protein resulting in Fluorescence
-
Cyclization step to form 5-membrane ring
- Gly-N –> attacks Ser amide carbonyl
-
Dehydration step
- forms unsaturated imidazoline
- DeHydrogenation of TYR carbons
- by OXYGEN to from conjugated fluorophore which:
- absorbs & emits light in th visible range
- by OXYGEN to from conjugated fluorophore which:
