Light interacting with matter as an analytical tool

  Light interacting with matter as an analytical tool Electronic Excitation by UV/Vis Spectroscopy :

  UV:

  Radio waves:

  IR: X-ray:

  valance

  Nuclear spin states molecular core electron

  electronic

  (in a magnetic feldd excitation vibrations

  excitation Where in the spectrum are these transitions?

  

Spectroscopic Techniques and

Chemistry they Probe

UV-vis UV-vis region bonding electrons

Atomic Absorption UV-vis region atomic transitions (val. e-)

FT-IR

  IR/Microwave vibrations, rotations Raman

  IR/UV vibrations FT-NMR Radio waves nuclear spin states

X-Ray Spectroscopy X-rays inner electrons, elemental

X-ray Crystallography X-rays 3-D structure

  Spectroscopic Techniques and Common Uses UV-vis UV-vis region

  Quantitative analysis/Beer’s Law Atomic Absorption UV-vis region

  Quantitative analysis Beer’s Law FT-IR

  IR/Microwave Functional Group Analysis Raman

  IR/UV Functional Group Analysis/quant

  FT-NMR Radio waves Structure determination X-Ray Spectroscopy X-rays Elemental Analysis X-ray Crystallography X-rays 3-D structure Anaylysis

Different Spectroscopies

  UV-vis – electronic states of valence e/d-

• orbital transitions for solvated transition metals Fluorescence – emission of UV/vis by certain
• molecules

  FT-IR – vibrational transitions of molecules

• FT-NMR – nuclear spin transitions

• X-Ray Spectroscopy – electronic transitions

• of core electrons

  Why should we learn this stuf?

After all, nobody solves structures with

UV any longer!

  Many organic molecules have chromophores that absorb UV UV absorbance is about 1000 x easier to detect per mole than NMR Still used in following reactions where the chromophore changes. Useful because timescale is so fast, and sensitivity so high. Kinetics, esp. in biochemistry, enzymology.

Most quantitative Analytical chemistry in organic chemistry is conducted using

HPLC with UV detectors One wavelength may not be the best for all compound in a mixture.

Uses for UV, continued

  

Knowing UV can help you know when to be skeptical of quant results. Need to

calibrate response factors Assessing purity of a major peak in HPLC is improved by “diode array” data,

taking UV spectra at time points across a peak. Any diferences could suggest a

unresolved component. “Peak Homogeneity” is key for purity analysis. Sensitivity makes HPLC sensitive e.g. validation of cleaning procedure for a production vessel

But you would need to know what compounds could and could not be detected

by UV detector! (Structure!!!d

One of the best ways for identifying the presence of acidic or basic groups, due

to big shifts in  for a chromophore containing a phenol, carboxylic acid, etc.

  “hypsochromic” shift “bathochromic” shift

The UV Absorption process

•   * and   * transitions: high-energy, accessible in vacuum UV ( <150 nmd. Not usually _max observed in molecular UV-Vis.
• n  * and   * transitions: non-bonding electrons (lone pairsd, wavelength ( d in the 150-250 _max nm region.
• n  * and   * transitions: most common transitions observed in organic molecular UV-Vis, observed in compounds with lone pairs and multiple bonds with  = 200-600 nm. _max
• Any of these require that incoming photons match in energy the gap corrresponding to a transition from

  Example

  π* π* π*

  What are the for a

  π* π* π*

  simple

  π* π* π*

  enone nature of these

  n n n

  π π π

  absorptions?

  π π

  π _{ }  =218 =320 _{max max}

• *; n-*;

   =11,000  =100

  

Example:   * transitions responsible for ethylene UV absorption at ~170 nm

calculated with ZINDO semi-empirical excited-states methods (Gaussian 03Wd: 170nm photon

  h How Do UV spectrometers work?

  Rotates, to achieve

  Matched quartz cuvettes

  scan

• _-5 Sample in solution at ca. 10 M.

  System protects PM tube from stray light D2 lamp-UV Tungsten lamp-Vis Double Beam makes it a _{voltage drives amplifer.}

_{inputs, diferential}

_{Two photomultiplier}

diference technique

Diode Array Detectors

  Diode array alternative puts grating, array of photosens. Semiconductors after the light goes through the sample. Advantage, speed, sensitivity, The Multiplex advantage Disadvantage,

  Model from Agilent literature. Imagine

  resolution is 1 nm, vs

Experimental details

  What compounds show UV spectra? Generally think of any unsaturated compounds as good candidates. Conjugated double bonds are strong absorbers Just heteroatoms are not enough but C=O are reliable Most compounds have “end absorbance” at lower frequency.

  Unfortunately solvent cutofs preclude observation. You will fnd molar absorbtivities  in L•cm/mol, tabulated. Transition metal complexes, inorganics Solvent must be UV grade (great sensitivity to impurities with double bondsd

  An Electronic Spectrum Make solution of concentration low enough that A≤ 1 (Ensures Linear Beer’s law behaviord

  1.0 Even though a dual beam  _max with certain

  goes through a solvent blank, choose solvents

  UV Visible extinction 

  that are UV transparent. Can extract the  value if conc. (Md and b (cmd are e known nc

  UV bands are much ba broader than the photonic or transition event. This is because vibration levels bs are superimposed on UV.

  A

  0.0

Solvents for UV (showing high energy cutofsd

  Water 205 CH ₃ CN 210

  C ₆ H ₁₂ 210 Ether 210 EtOH 210 Hexane 210 MeOH 210

  THF 220 CH ₂ Cl ₂ 235 CHCl ₃ 245

  CCl ₄ 265 benzene 280 Acetone 300 Various bufers for

Organic compounds (many of themd have UV spectra

  One thing is clear Uvs can be very non-specifc Its hard to interpret except at a cursory level, and to say that the spectrum is consistent with the structure Each band can be a superposition of many transitions Generally we don’t assign the An Example--Pulegone

  Frequently plotted as

  log of

  molar extinction _O

   So at 240 nm, pulegone has a molar

  Can we calculate UVs? _{Elec tronic Spectra} Wavelength (nm) ^{Molar Absorptivity (l/mol-cm)} _{220 230 240 250 260 270 280 290 300}

¹⁰⁰⁴⁹

²⁰⁰⁹⁷

³⁰¹⁴⁶

⁴⁰¹⁹⁴

⁵⁰²⁴³

_{Electronic Spectra} ^nacindolA _{Molar Absorptivity (l/mol-cm)}

  

⁵¹⁹⁷²

⁴¹⁵⁷⁸

20789

³¹¹⁸³

  Semi-empirical (MOPACd at AM1, then ZINDO for confg. interaction level 14

  The orbitals involved Electronic Spectra _{Molar Absorptivity (l/mol-cm)}

  ^{55487 44390 33292} 11097 ²²¹⁹⁵

  Showing atoms whose MO’s contribute The Quantitative Picture Transmittance:

• P P (power outd (power ind

  T = P/P Absorbance:

• A = -log T = log P /P

  10

  The Beer-Lambert Law (a.k.a. Beer’s Lawd:

• A = ebc _{e is the molar absorbtivity with units of L mol cm} Where the absorbance A has no units, since A = log P / P _{-1 -1}
₁₀

Beer-Lambert Law

  Linear absorbance with increased concentration--directly proportional Makes UV useful for quantitative analysis and in HPLC detectors Above a certain concentration the linearity curves down, loses direct proportionality--Due to molecular associations at higher concentrations. Must demonstrate linearity in validating response in an analytical procedure.

  Quantitative Spectroscopy Beer’s Law

• A = e bc

  l1 l1

e is molar absorptivity (unique for a

given compound at l )

  1 b is path length c concentration Beer’s Law cuvette slit source detector

  A = -logT = log(P /P) = ebc

• T = P /P = P/P
• solution solvent

  Works for monochromatic light

  Characteristics of Beer’s Law Plots One wavelength

• Good plots have a range of
• absorbances from 0.010 to 1.000 Absorbances over 1.000 are not that
>valid and should be avoided 2 orders of magnitude

Standard Practice

  Prepare standards of known

• concentration Measure absorbance at lmax
• Plot A vs. concentration
• Obtain slope
• Use slope (and intercept) to determine
• the concentration of the analyte in the unknown

Typical Beer’s Law Plot

  1.2

  1 y = 0.02x

  0.8

  0.4

  0.2

  0.0

  20.0

  40.0

  60.0 concentration (uM) UV-Vis Spectroscopy

• UV- organic molecules
• – Outer electron bonding transitions
• – conjugation
• Visible – metal/ligands in solution
• – d-orbital transitions
• Instrumentation

Characteristics of UV-Vis spectra of Organic Molecules

  Absorb mostly in UV unless highly

• conjugated Spectra are broad, usually to broad for
• qualitative identification purposes Excellent for quantitative Beer’s Law-
• type analyses The most common detector for an
• HPLC

Molecules have quantized energy levels: ex. electronic energy levels.

  hv gy gy er er en en

   }

  = hv

  Q: Where do these quantized energy levels come from? A: The electronic confgurations associated with bonding.

  Each electronic energy level (confgurationd has associated with it the many vibrational energy levels we

  Broad spectra Overlapping vibrational and rotational

• peaks Solvent effects

  Molecular Orbital Theory

• Fig 18-10

  2s 2s s

• ^* s s ^* p ^* p

  2p 2p n

  C C

  Ethane

    _hv  

   _{H H H C C H}  _H  _{H max} = 135 nm (a high energy transitiond

Absorptions having  < 200 nm are difcult to observe because

_max

  _{C C}   

    _hv = hv =hc/

    

    

  Example: ethylene absorbs at longer wavelengths:  _max = 165 nm = 10,000 C O 

     _n hv _n 

   

_n

 

  The n to pi* transition is at even lower wavelengths but is not as strong as pi to pi* transitions. It is said to be “forbidden.” Example:

  C C

   _{C C} 135 nm

   _H 165 nm

  C O

  n183 nm weak _{C O}  150 nm n188 nm n279 nm weak

  180 nm C O

  A Conjugated systems: _{C C HOMO} LUMO _{(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).} Preferred transition is between Highest Occupied Molecular Orbital Note: Additional conjugation (double bondsd lowers the HOMO- LUMO energy gap: Example:

  O O ^O

  Similar structures have similar UV spectra:

   _max = 238, 305 nm ^{ max}

= 240, 311 nm

_{ max} = 173, 192 nm

  Lycopene:  _max = 114 + 5(8d + 11*(48.0-1.7*11d = 476 nm _{ max} (Actuald = 474.

  Polyenes, and Unsaturated Carbonyl groups; an Empirical triumph

  R.B. Woodward, L.F. Fieser and others Predict  for π  * in extended conjugation _max systems to within ca. 2-3 nm.

  Attached group increment, nm Extend conjugation +30 Homoannular, base 253 nm Addn exocyclic DB +5

  Alkyl +5 O-Acyl Acyclic, base 217 nm S-alkyl +30

  O-alkyl +6 Similar for Enones

  O x  b b ^{O O}

  X=H 207 X=R 215 X=OH 193 X=OR 193

  215 202 227 239

  Base Values, add these increments… Extnd C=C +30 Add exocyclic C=C

   b g ,+ d

• 5 Homoannular diene +39 alkyl +10 +12 +18 +18 OH +35 +30 +50 OAcyl +6 +6 +6 +6 O-alkyl +35 +30 +17 +31 NR
₂ S-alkyl

  With solvent correction _{of….. Water +8} EtOH 0 _{CHCl 3 -1}

  Some Worked Examples Base value 217 2 x alkyl subst. 10 exo

DB 5 total

232 Obs. 237

  Base value 214 3 x alkyl subst. 15 exo DB 5 total

234 Obs. 235

  O Base value 215 2 ß alkyl subst. 24 Distinguish Isomers! Base value 214 4 x alkyl subst. 20 exo DB 5 total 239 Obs. 238

  HO C ₂ Base value 253 4 x alkyl subst. 20 total 273 Obs. 273

Generally, extending conjugation leads to red shift

  “particle in a box” QM theory; bigger box Substituents attached to a chromophore that cause a red shift are called “auxochromes” Strain has an efect…

   253 239 256 248

Interpretation of UV-Visible Spectra

  Transition metal

• complexes; d, f electrons. Lanthanide • complexes – sharp lines caused by “screening” of the f electrons by other orbitals One advantage of this
• is the use of holmium oxide flters (sharp linesd for wavelength

  UV of Benzene in heptane ^{Group K band () B band}

  Benzenoid aromatics

  () ^{R band} _{Alkyl 208(7800d 260(220d --}
• _{-OH 211(6200d 270(1450d}

• -O

  ^{- 236(9400d 287(2600d} _-OCH
_{3 217(6400d 269(1500d} NH
• _{-F 204(6200d 254(900d 2} ^{230(8600d 280(1400d} _{-Cl 210(7500d 257(170d}
• Br 210(7500d 257(170d _{-I 207(7000d 258/285(610/180 d}
• NH
• _{-C=CH 3} ^{+ 203(7500d 254(160d}
₂ 248(15000d 282(740d<
• CCH 248(17000d 278(6500

  _-C

_{6 H}
• _{-C(=OdH 242(14000d 280(1400d 328(55d 6 250(14000d}
• C(=OdR 238(13000d 276(800d 320(40d _-CO
_{2 H 226(9800d 272(850d}
Substituent efects don’t really add up Can’t tell any thing about substitution geometry Exception to this is when adjacent substituents can interact, e.g hydrogen bonding.

  E.g the secondary benzene band at 254 shifts to 303 in salicylic acid In p-hydroxybenzoic acid, it is at the phenol or benzoic acid frequency

Heterocycles

  Nitrogen heterocycles are pretty similar to the benzenoid anaologs that are isoelectronic.

  Can study protonation, complex formation (charge transfer bandsd

  Quantitative analysis

  Great for non- aqueous titrations Example here gives detn of endpoint for

  Isosbestic points

  bromcresol green

  Single clear point, can exclude

  Binding studies

  intermediate state, exclude light scattering and Beer’s law applies

  Form I to form II More Complex Electronic Processes Fluorescence: absorption of

• radiation to an excited state, followed by emission of radiation to a lower state of the same multiplicity

  Phosphorescence: absorption of

• radiation to an excited state, followed by emission of radiation to a lower state of diferent multiplicity

  Singlet state: spins are paired,

• no net angular momentum (and no net magnetic feldd

Metal ion transitions

  Degenerate D-orbitals of naked Co

  D-orbitals of hydrated Co ²⁺ D E Octahedral Geometry

  H O ₂ H O ₂ H O ₂

  

2+

  Co H O ₂ H O ₂ H O ₂

Instrumentation

• Fixed wavelength instruments
• Scanning instruments
• Diode Array Instruments

Fixed Wavelength Instrument

  LED serve as source

• Pseudo-monochromatic light source
• No monochrometer necessary/ wavelength selection
• occurs by turning on the appropriate LED

  4 LEDs to choose from

• sample beam of light

  LEDs photodyode

Scanning Instrument

  cuvette Tungsten Filament (vis) slit

  Photomultiplier tube monochromator

  Deuterium lamp slit Scanning Instrument sources

• Tungten lamp (350-2500 nm)
• Deuterium (200-400 nm)
• Xenon Arc lamps (200-1000 nm)

  

Monochromator

Braggs law, nl = d(sin i + sin r)
• Angular dispersion , dr/dl = n / d(cos rd
• Resolution, R Dl nN, resolution is = l/ =
• extended by concave mirrors to refocus the divergent beam at the exit slit

Sample holder

• Visible; can be plastic or glass
• UV; you must use quartz

  

Single beam vs. double beam

• Source flicker

Diode array Instrument

  mirror Diode array detector 328 individual detectors

  Tungsten Filament (vis) slit slit cuvette Deuterium lamp

  Filament (UV)

  Advantages/disadvantages Scanning instrument

• High spectral resolution (63000), l/Dl
• –

  Long data acquisition time (several

• – minutes) Low throughput
• – Diode array
• Fast acquisition time (a couple of
• – seconds), compatible with on-line separations High throughput (no slits)
• –

HPLC-UV

  Mobile phase HPLC Pump syringe 6-port valve Sample loop

  HPLC column UV detector