20/21 - Spectroscopy

Question 1

Some uses of SPECTROSCOPY:

Answer 1

Mixture of compounds -> Chromatography -> single compound

Single Compound -> KNOWLEDGE OF STRUCTURE

Bio System + Compound –> Compound Localization / MoA / Protein-protein Interactions

Question 2

What is SPECTROSCOPY?

Answer 2

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

Question 3

4 First (smallest) Wavelength Regions

& what they measure

Answer 3

Gamma rays - n/a

X-Rays - core/inner level electrons

UV & Visible** = **valence / outer electrons

Question 4

3 LAST (largest) Wavelength Regions

& what they measure

Answer 4

IR** **- molecular VIBRATIONS

Microwave - molecular ROTATIONS

Radio - NUCLEAR SPIN

Question 5

What does a spectrum measure?

Answer 5

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

Question 6

Typical process of “small molecule” structure ELUCIDATION

Answer 6

Compound ->

IR / UV-Vis / MS ->

1D NMR / 2DNMR ->

IDENTIFIED STRUCTURE

X-Ray Cytallography SKIPS all those steps

Question 7

X-Ray Crystallography

Energy Interaction / Central Concept

Answer 7

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

Question 8

X-Ray Crystallography

Used for / Works on?

Answer 8

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

Question 9

X-Ray Crystallography

strengths & weaknesses

Answer 9

• 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

Question 10

IR (infrared) Spectroscopy

Energy interaction / Central Concept

Answer 10

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

Question 11

IR (infrared) Spectroscopy

Used for / Works For?

Answer 11

presence of FUNCTIONAL GROUPS

Bond Distance

Natural product elucidation, but NMR/MASS spectroscopy is better

need to compare to a reference / cheat sheet

Question 12

IR (infrared) Spectroscopy

Strength / Weakness

Answer 12

• Strength:
  
  • ​Indicates potential presence of FUNCTIONAL GROUPS
    
    • including bond distance
• 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

Question 13

Atomic Absorption (aa) Spectroscopy

Energy Interaction / Central Concept

Answer 13

EMISSION SPECTRA of each element produces a unique fingerprint

Flame Test - similar to FP (Flame Photometry)

Flame converts metal ions -> atoms

measures Light Intensity

Question 14

AA Spectroscopy

Works on / Common uses

Answer 14

Determination of METALS (Hg / Pb / etc)

from LIQUID** **(urine / plasma / serum)

Question 15

AA Spectroscopy

Strengths / Weaknesses

Answer 15

• Strength:
  
  • ​__used for Metals in Liquids/Solution
• ​Weakness:
  
  • ​Low throughput, requires manual operation
    
    • ​_DESTRUCTIVE_

Question 16

UV-Vis Spectroscopy

Energy Interaction / Central Concept

Answer 16

• Spec of UV & Vis regions measures valence / outer electrons​
  
  • Molecules absorb EM radiation
    
    • as atoms pass from ground state -> Excited State
• ​More effectively a molecule ABSORBS LIGHT (given wavelength,
  
  • –> GREATER the extent of light ABSORBANCE
• views CHROMOPHORE
  
  • functional group that absorbs UV radiation

Question 17

Chromophore

Answer 17

Functional group that absorbs UV Radiation

views CONJUGATION

organic molecules between 190-800 nm

detected by UV / PDA Detectors

Question 18

Name the Process of rapidly identifying compounds that are

KNOWN CHEMICAL SCAFFOLDS

Answer 18

DEREPLICATION

used in UV/PDA

Question 19

UV / PDA Detectors

PhotoDiode Array

Answer 19

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

Question 20

UV / PDA Spectroscopy

S / W

Answer 20

• 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

Question 21

Common Chromophores

Answer 21

Blue box = solvent interference

chromophore wavelength OVERLAPS

w/ wavelength of solvent

Question 22

Chromophres with more complex conjugation

Answer 22

Complex conjugation = more DISTINCT UV Fingerprint

Question 23

UV / PDA Spectroscopy

Works on / Common uses

Answer 23

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

Question 24

ELSD

Evaporative Light Scattering Dector

Concept / Detects for?

Answer 24

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

Question 25

**ELSD** **Evaporative Light Scattering Dector** Steps / Method

Answer 25

***_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

Question 26

**ELSD Detection** **Used For?**

Answer 26

detects **Physical Presence** useful for detecting molecules that *do* ***NOT have any spectroscopic qualities*** *is destructive though*

Question 27

**Fluorescense vs Absorbance**

Answer 27

*You can't have **_fluorescence_ without absorbance*** * **Absorbance** * FIRST step in _fluorescence_. * electronic transition that promotes electron from ground state (LIGHT ENERGY) * **_Fluorescence_** * the **light emitted** by an atom or molecule * after absorbance of EM energy

Question 28

**Fluoresence**

Answer 28

* **_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

Question 29

**Fluorophores**

Answer 29

* _Molecules or Functional groups_ that have the * **_Capacity to exhibit flourescence_** * ​Structural characteristic of useful fluorophores: * **Extended Conjugated Pi bonds** * the **MORE** **conj bonds** * & *l**ower the excited energy requirement*** * ***​*****_LONGER the wavelength (redder) the exciting light can be_** * **_​​_**Ex. **GFP**, green fluorescent protein

Question 30

**_GFP Mechanism_**

Answer 30

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