UV VISIBLE SPECTROSCOPY

UV & electronic transitions Usable ranges & observations Band Structure Instrumentation & Spectra Beer-Lambert Law Application of UV-spec Calibration

BY NANDESH V. PINGALE.

Electromagnetic Radiation
Radiation is absorbed & emmited in photons. The defining characterstic of a photon is that its energy cannot be split into smaller pieces. Each photons energy is defined by its frequency () or wave lenth () or wavenumber (wn) Ephoton = h = hc/ = hc (wn) Two constants appear in these formulas h = planks constant, 6.63 x 10-34 J s c = speed of light, 3.00 x 108 m s-1 Wave number (wn) = 1/

UV Spectroscopy

I.

Introduction A. UV radiation and Electronic Excitations 1. The difference in energy between molecular bonding, non-bonding and anti-bonding orbitals ranges from 125-650 kJ/mole 2. 3. This energy corresponds to EM radiation in the ultraviolet (UV) region, 100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum For comparison, recall the EM spectrum: X-rays UV

-rays

IR

Microwave

Radio

Visible

4. 5.

Using IR we observed vibrational transitions with energies of 8-40 kJ/mol at wavelengths of 2500-15,000 nm For purposes of our discussion, we will refer to UV and VIS spectroscopy as UV

UV Spectroscopy

I.

Introduction B. Observed electronic transitions 1. The lowest energy transition (and most often obs. by UV) is typically that of an electron in the Highest Occupied Molecular Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital (LUMO) 2. For any bond (pair of electrons) in a molecule, the molecular orbitals are a mixture of the two contributing atomic orbitals; for every bonding orbital created from this mixing (s, p), there is a corresponding antibonding orbital of symmetrically higher energy (s*, p*) The lowest energy occupied orbitals are typically the s; likewise, the corresponding anti-bonding s* orbital is of the highest energy p-orbitals are of somewhat higher energy, and their complementary antibonding orbital somewhat lower in energy than s*. Unshared pairs lie at the energy of the original atomic orbital, most often this energy is higher than p or s (since no bond is formed, there is no benefit in energy)

3. 4. 5.

UV Spectroscopy I. Introduction B. Observed electronic transitions 6. Here is a graphical representation

s* p* Energy Atomic orbital

Unoccupied levels

Atomic orbital Occupied levels

p s Molecular orbitals

UV Spectroscopy I. Introduction B. Observed electronic transitions 7. From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy:

Energy

alkanes carbonyls unsaturated cmpds. O, N, S, halogens carbonyls

n n

UV Spectroscopy I. Introduction B. Observed electronic transitions 7. Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not transparent below 200 nm 8. 9. Special equipment to study vacuum or far UV is required Routine organic UV spectra are typically collected from 200-700 nm

10. This limits the transitions that can be observed: alkanes carbonyls unsaturated cmpds. O, N, S, halogens carbonyls 150 nm 170 nm 180 nm 190 nm 300 nm - if conjugated!

n n

UV Spectroscopy I. Introduction E. Band Structure 1. Unlike IR (or later NMR), where there may be upwards of 5 or more resolvable peaks from which to elucidate structural information, UV tends to give wide, overlapping bands 2. It would seem that since the electronic energy levels of a pure sample of molecules would be quantized, fine, discrete bands would be observed for atomic spectra, this is the case In molecules, when a bulk sample of molecules is observed, not all bonds (read pairs of electrons) are in the same vibrational or rotational energy states This effect will impact the wavelength at which a transition is observed very similar to the effect of H-bonding on the O-H vibrational energy levels in neat samples

3.

4.

UV Spectroscopy II. Instrumentation and Spectra A. Instrumentation 1. The construction of a traditional UV-VIS spectrometer is very similar to an IR, as similar functions sample handling, irradiation, detection and output are required 2. Here is a simple schematic that covers most modern UV spectrometers: log(I0/I) = A sample
UV-VIS sources

I0

I detector
200 , nm 700

I0

reference

monochromator/ beam splitter optics

I0

UV Spectroscopy II. Instrumentation and Spectra A. Instrumentation 3. Two sources are required to scan the entire UV-VIS band: Deuterium lamp covers the UV 200-330 Tungsten lamp covers 330-700 3. As with the dispersive IR, the lamps illuminate the entire band of UV or visible light; the monochromator (grating or prism) gradually changes the small bands of radiation sent to the beam splitter The beam splitter sends a separate band to a cell containing the sample solution and a reference solution The detector measures the difference between the transmitted light through the sample (I) vs. the incident light (I0) and sends this information to the recorder

4. 5.

UV Spectroscopy II. Instrumentation and Spectra A. Instrumentation 7. As with dispersive IR, time is required to cover the entire UV-VIS band due to the mechanism of changing wavelengths 8. A recent improvement is the diode-array spectrophotometer - here a prism (dispersion device) breaks apart the full spectrum transmitted through the sample Each individual band of UV is detected by a individual diodes on a silicon wafer simultaneously the obvious limitation is the size of the diode, so some loss of resolution over traditional instruments is observed
Diode array UV-VIS sources

9.

sample

Polychromator entrance slit and dispersion device

UV Spectroscopy II. Instrumentation and Spectra A. Instrumentation Sample Handling 1. Virtually all UV spectra are recorded solution-phase 2. 3. 4. Cells can be made of plastic, glass or quartz Only quartz is transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra Concentration (we will cover shortly) is empirically determined

A typical sample cell (commonly called a cuvet):

UV Spectroscopy II. Instrumentation and Spectra C. The Spectrum 1. The x-axis of the spectrum is in wavelength; 200-350 nm for UV, 200-700 for UV-VIS determinations 2. Due to the lack of any fine structure, spectra are rarely shown in their raw form, rather, the peak maxima are simply reported as a numerical list of lamba max values or max

NH2

max =
O O

206 nm 252 317 376

UV Spectroscopy II. Instrumentation and Spectra C. The Spectrum 1. The y-axis of the spectrum is in absorbance, A 2. From the spectrometers point of view, absorbance is the inverse of transmittance: A = log10 (I0/I) From an experimental point of view, three other considerations must be made: i. a longer path length, l through the sample will cause more UV light to be absorbed linear effect ii. iii. the greater the concentration, c of the sample, the more UV light will be absorbed linear effect some electronic transitions are more effective at the absorption of photon than others molar absorptivity, this may vary by orders of magnitude

3.

UV Spectroscopy II. Instrumentation and Spectra C. The Spectrum 4. These effects are combined into the Beer-Lambert Law: i. ii. iii.

A = c l

for most UV spectrometers, l would remain constant (standard cells are typically 1 cm in path length) concentration is typically varied depending on the strength of absorption observed or expected typically dilute sub .001 M molar absorptivities vary by orders of magnitude: values of 104-106 104-106 are termed high intensity absorptions values of 103-104 are termed low intensity absorptions values of 0 to 103 are the absorptions of forbidden transitions

A is unitless, so the units for are cm-1 M-1 and are rarely expressed 5. Since path length and concentration effects can be easily factored out, absorbance simply becomes proportional to , and the y-axis is expressed as directly or as the logarithm of

UV Spectroscopy II. Instrumentation and Spectra D. Practical application of UV spectroscopy 1. UV was the first organic spectral method, however, it is rarely used as a primary method for structure determination 2. 3. It is most useful in combination with NMR and IR data to elucidate unique electronic features that may be ambiguous in those methods It can be used to assay (via max and molar absorptivity) the proper irradiation wavelengths for photochemical experiments, or the design of UV resistant paints and coatings The most ubiquitous use of UV is as a detection device for HPLC; since UV is utilized for solution phase samples vs. a reference solvent this is easily incorporated into LC design

4.

UV is to HPLC what mass spectrometry (MS) will be to GC

UV Spectroscopy III. Chromophores A. Definition 1. Remember the electrons present in organic molecules are involved in covalent bonds or lone pairs of electrons on atoms such as O or N 2. Since similar functional groups will have electrons capable of discrete classes of transitions, the characteristic energy of these energies is more representative of the functional group than the electrons themselves A functional group capable of having characteristic electronic transitions is called a chromophore (color loving) Structural or electronic changes in the chromophore can be quantified and used to predict shifts in the observed electronic transitions

3. 4.

UV Spectroscopy III. Chromophores B. Organic Chromophores 1. Alkanes only posses -bonds and no lone pairs of electrons, so only the high energy * transition is observed in the far UV This transition is destructive to the molecule, causing cleavage of the bond
C C

UV Spectroscopy III. Chromophores B. Organic Chromophores 3. Alkenes and Alkynes in the case of isolated examples of these compounds the * is observed at 175 and 170 nm, respectively Even though this transition is of lower energy than *, it is still in the far UV however, the transition energy is sensitive to substitution

UV Spectroscopy III. Chromophores B. Organic Chromophores 4. Carbonyls unsaturated systems incorporating N or O can undergo * transitions (~285 nm) in addition to *

Despite the fact this transition is forbidden by the selection rules ( = 15), it is the most often observed and studied transition for carbonyls This transition is also sensitive to substituents on the carbonyl Similar to alkenes and alkynes, non-substituted carbonyls undergo the * transition in the vacuum UV (188 nm, = 900); sensitive to substitution effects

UV Spectroscopy III. Chromophores B. Organic Chromophores 4. Carbonyls n * transitions (~285 nm); * (188 nm)

It has been determined from spectral studies, that carbonyl oxygen more approximates sp rather than sp2 !

CO transitions omitted for clarity

UV Spectroscopy III. Chromophores A. Substituent Effects General from our brief study of these general chromophores, only the weak n * transition occurs in the routinely observed UV The attachment of substituent groups (other than H) can shift the energy of the transition Substituents that increase the intensity and often wavelength of an absorption are called auxochromes Common auxochromes include alkyl, hydroxyl, alkoxy and amino groups and the halogens

UV Spectroscopy III. Chromophores A. Substituent Effects General Substituents may have any of four effects on a chromophore i. Bathochromic shift (red shift) a shift to longer ; lower energy i. ii. iii. Hypsochromic shift (blue shift) shift to shorter ; higher energy Hyperchromic effect an increase in intensity Hypochromic effect a decrease in intensity
Hyperchromic

Hypsochromic

Bathochromic

Hypochromic

200 nm

700 nm

UV Spectroscopy III. Chromophores A. Substituent Effects 1. Conjugation most efficient means of bringing about a bathochromic and hyperchromic shift of an unsaturated chromophore:
H2C CH2

max nm
175 217 258

15,000 21,000 35,000

465
-carotene
O

125,000

n * 280 * 189 n * 280 * 213

12 900 27 7,100

UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 1. General Features Although aromatic rings are among the most widely studied and observed chromophores, the absorptions that arise from the various electronic transitions are complex On first inspection, benzene has six -MOs, 3 filled , 3 unfilled *

6 4 5

2 1

UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 1. General Features One would expect there to be four possible HOMO-LUMO * transitions at observable wavelengths (conjugation) Due to symmetry concerns and selection rules, the actual transition energy states of benzene are illustrated at the right:

6 4 5
200 nm (forbidden)

1 u 1 u 2 u
180 nm (allowed)

2 1

260 nm (forbidden)

1 g

UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 1. General Features The allowed transition ( = 47,000) is not in the routine range of UV obs. at 180 nm, and is referred to as the primary band The forbidden transition ( = 7400) is observed if substituent effects shift it into the obs. region; this is referred to as the second primary band At 260 nm is another forbidden transition ( = 230), referred to as the secondary band. This transition is fleetingly allowed due to the disruption of symmetry by the vibrational energy states, the overlap of which is observed in what is called fine structure

UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 1. General Features Substitution, auxochromic, conjugation and solvent effects can cause shifts in wavelength and intensity of aromatic systems similar to dienes and enones However, these shifts are difficult to predict the formulation of empirical rules is for the most part is not efficient (there are more exceptions than rules) There are some general qualitative observations that can be made by classifying substituent groups --

UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects a. Substituents with Unshared Electrons If the group attached to the ring bears n electrons, they can induce a shift in the primary and secondary absorption bands Non-bonding electrons extend the -system through resonance lowering the energy of transition * More available n-pairs of electrons give greater shifts
G G G

UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects a. Substituents with Unshared Electrons The presence of n-electrons gives the possibility of n * transitions If this occurs, the electron now removed from G, becomes an extra electron in the anti-bonding * orbital of the ring This state is referred to as a charge-transfer excited state
G G * * G *

UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects d. Di-substituted and multiple group effects With di-substituted aromatics, it is necessary to consider both groups If both groups are electron donating or withdrawing, the effect is similar to the effect of the stronger of the two groups as if it were a mono-substituted ring If one group is electron withdrawing and one group electron donating and they are para- to one another, the magnitude of the shift is greater than the sum of both the group effects Consider p-nitroaniline:
O H2N N O H2N N O O

UV Spectroscopy V. Visible Spectroscopy A. Color 1. General The portion of the EM spectrum from 400-800 is observable to humans- we (and some other mammals) have the adaptation of seeing color at the expense of greater detail

400

500

600

700

800

, nm Violet Indigo Blue Green Yellow Orange Red 400-420 420-440 440-490 490-570 570-585 585-620 620-780

UV Spectroscopy V. Visible Spectroscopy A. Color 1. General When white (continuum of ) light passes through, or is reflected by a surface, those ls that are absorbed are removed from the transmitted or reflected light respectively What is seen is the complimentary colors (those that are not absorbed) This is the origin of the color wheel

UV Spectroscopy V. Visible Spectroscopy A. Color 1. General Organic compounds that are colored are typically those with extensively conjugated systems (typically more than five) Consider -carotene

-carotene, max = 455 nm

max is at 455 in the far blue region of the spectrum this is absorbed The remaining light has the complementary color of orange

UV Spectroscopy V. Visible Spectroscopy A. Color 1. General These materials are some of the more familiar colors of our environment
NO2

HO
N N OH

H2N

N N NH2

O3S

N N

SO3
Para Red

Fast Brown

Sunset Yellow (Food Yellow 3)

UV Spectroscopy V. Visible Spectroscopy A. Color 1. General In the chemical sciences these are the acid-base indicators used for the various pH ranges:
Methyl Orange CH3 N CH3 CH3 N CH3

Remember the effects of pH on aromatic substituents

O3S

N N

O3S

H N N

Yellow, pH > 4.4

Red, pH < 3.2

ANY QUESTIONS