UV/Vis (Ultraviolet and Visible) Spectroscopy

Agilent 8453 Diode Array UV-Vis Spectrophotometer

Varian Cary 5000 UV-Vis-NIR Spectrophotometer

To Do’s •  Read Chapters 13 & 14. •  Complete the end-of-chapter problems, 13-1, 13-2, 13-3,13-6 and 13-8. •  Complete the end-of-chapter problems, 14-2, 14-5, 14-6, 14-7, 14-8, 14-9, 14-10, 14-12, 14-14, 14-16, 14-19 and 14-20.

Wavelength and Spectroscopy

What Molecular Changes Cause UV/Vis Absorptions?

λ = 200 ~ 700 nm

UV/Vis spectroscopy is used to detect the presence of certain functional groups called chromophores, especially in conjugate systemssuch as aromatics, dienes, polyenes, and α,β -unsaturated ketones

Electronic excitation

Absorption of UV or visible light occurs only when the energy of incident radiation is the same as that of an electronic transition in the molecule.

Electronic Transitions

Only n π* and π in 200 - 700 nm.

π* transitions usually show up

Only limited set of organic functional groups show UV/Vis absorptions.

Instrument (double beam)

Typical λ = 200 ~ 700 nm

The Beer’s Law Aλ = log (

Iref Isample

)

Iref Isample

> 1, thus Aλ is aways positive. Iref

Aλ = ελ x c x l

Isample Iref Isample

ελ : molar absorptivity constant or extinction coefficient (l mol-1 cm-1)

c : concentration (mol/l) l : path length (cm)

Iref Isample Iref Isample

=1

Aλ = 0

= 10

Aλ = 1

= 100

Aλ = 2

= 1000

Aλ = 3

Instrument (Diode Array)

~ one diode per 0.5 nm

Rugged, fast, and reliable

Solvents and Cuvettes for UV/Vis Spectroscopy

The Franck-Condon Principle

Nuclear motion is negligible during the time required for an electronic transition. Excitation can occur from the ground state to any of the excited state vibrational levels.

A

B The lines overlap and a continuous broad band is observed.

Typical UV/Vis Spectra O

CH3

Due to the lack of any fine structure, spectra are rarely shown in their raw form, rather, the peak maxima(λmax) and their εmax are commonly reported.

Isosbestic Points

εHA = εA-

HA

A-

Isosbestic points are usually a diagnostic for the presence of only two absorbing species.

Spectral Shift

blue shift

red shift

Polymeric Nucleic Acids Have an Increased Molar Absorptivity When They are Converted to Smaller Units

Difference Spectroscopy

Solid line minus dotted line

UV absorption spectra and temperature-difference spectra of HO5dCyt in 0.02 M NaOH/ NaHCO3 buffer at pH 10.5 at 25°C (A, dashed line), 75°C (A, solid line) and 75° − 25°C (B). Comparable spectra of dCyt in 0.02 M NaOH/NaHCO3 buffer at pH 7 at 25°C (C, dashed line), 75°C (C, solid line) and 75° − 25°C (D).

Useful in detecting small changes.

Common Organic Chromophores

conjugated and non-conjugated

The Carbonyl Groups λmax εmax

270 ~ 300 nm 10 ~ 100, n π*

O R

C

O

and R1

R

C

H

π* c

o

n

The two orbitals, n and π*, are orthogonal to each other and promotion of electron requires significant changes in geometry. The electronic transition must be coupled with vibration that allows some overlap between these orbitals.

Other Carbonyl Groups O H3C

C

CH3

(hexane)

λmax

εmax

274

15

π* O R

O H 3C

C

Cl

(hexane)

235

53

(water)

214

-

C

n X

O H3C

C

NH2

π*

O H3C

C

OEt

(water)

204

60 C=O n (X)

O H3C

C

OH

(ethanolr)

204

41

n

Solvent Effects on n-π*(C=O) C

OI

H

O R

π*

n

Aldehydes and Ketons

Usually not observed

250 ~ 300 nm Low εmax

Unconjugated Alkenes

Usually not observed

Conjugated Alkenes

Conjugation

λmax, εmax

Conjugation and Spectral Shifts

β-Carotene

Empirical Calculation of λmax for Conjugated Alkenes

Empirical Calculation - Examples

Empirical Calculation – More Examples

Diene Isomers λmax = 239 nm

λmax = 278 nm

7

6

5

4

7

6

5

4

PPM

PPM

3

2

1

0

3

2

1

0

Steric Hindrance and Spectral Shifts CH 3

H 3C

CH 3

CH3 CH3

CH3 H3 C

H 3C

H3 C

CH 2

λmax

225

231

εmax

6.400

10,000

CH 3 CH 3

CH3 H3 C

CH 2

Twisted diene CH 2

conjugation

λmax and εmax

Conjugated Carbonyls

Conjugation

λmax, εmax

alkyl or ring residues

Empirical Calculation

Empirical Calculation - Examples

Exocyclic Alkene Issue

O

215 12  β-alkyl 3 x 18 δ+ alkyl 2x5 exocyclic C=C 2 x 30 extra conjugation 351 nm Observed 348 nm

Keto-enol Tautomerization

In hexane λmax = 295 εmax = 50

In ethanol λmax = 255 εmax = 12,500

215 12 (β-alkyl) 30 (β-OH) 257nm

Benzene Chromophore Ε1u

π6*

π4*

π5*

200 nm (forbidden)

Β1u Β2u

π2

π3

260 nm (forbidden)

180 nm (allowed)

Α1g

π1

Benzene Bands Designation

Benzene Chromophore

Substituent Effects on Benzene Chromophore •  Substitution, 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)

Benzene Derivatives - Monosubstituted

pH Sensitive Derivatives O

O OH

OH

1L

OH-

O

O

OH-

1L

a

b

Substituent

λmax

ε

λmax

ε

-H

203.5

7,400

254

204

-OH

211

6,200

270

1,450

-O-

235

9,400

287

2,600

-NH2

230

8,600

280

1,430

-NH3+

203

7,500

254

169

-C(O)OH

230

11,600

273

970

-C(O)O-

224

8,700

268

560

pH Titration of Phenol Isosbestic point

i

n

Aλ = [i]εi + [n]εn

pKa = - log [H+] - log[n]/[i]

At a particular λ, [n]/[i] = (A – Ai)/(An – A)

pKa = pH – log[n]/[i]

Ka = [n][H+]/[i]

pKa = pH – log(A – Ai)/(An – A)

pKa Measurement

Aromatic Amino Acids R O

H3N O

Phe

R=

OH

Tyr

H N

Trp

Benzene Derivatives Disubstituted

The mono-substituted moiety with the largest red shift will dominate the absorption of a di-substituted benzene.

Para-substituted Benzenes

If one substituent is e-withdrawing and the other is e-donating, a significant red-shift is observed due to the extended conjugation.

Disubstituted Anilines

Disubstituted Benzaldehydes

Disubstituted Benzoic Acids

Empirical Calculation – Benzoyl Derivatives

Empirical Calculation – Examples

246 (ketone) + 3 (o-ring) + 2 (m-Br)

Br

O

λmax = 251nm obs = 253 nm

CO2H

HO HO OH

230 (acid) + 7 x 2 (m-OH) + 25 (p-OH) λmax = 269nm obs = 270 nm

Heterocyclic Aromatics

Steric Effect – Cis & Trans

Steric Effect – E & Z Z E

E

E

Z Z

λmax

328

εmax

56,200

313

299

30,600

29,500

Steric Effect - Biphenyls

Tautomerization

Polynuclear Aromatics

When the number of fused aromatic rings increases, the λ for the 1La and 1Lb bands also increase.

Charge Transfer(CT) Bands A+D

A

D

CT complex

hν

A

D

Azo Dyes • 

One of the most common class of colored organic molecules are the azo dyes: N N

EWGs

EDGs

Like di-subsituted aromatic chromophores, the effect of opposite groups is greater than the sum of the individual effects when they are placed on different rings. Coincidentally, it is necessary for these to be opposite for the original synthetic preparation!

58

M&M Chocolates

The colors of M&M’s Bright Blue

Royal Blue

Common Food Uses Beverages, dairy products, powders, jellies, confections, condiments, icing.

Common Food Uses Baked goods, cereals, snack foods, ice-cream, confections, cherries.

Orange-red

Lemon-yellow

Common Food Uses Gelatins, puddings, dairy products, confections, beverages, condiments.

Common Food Uses Custards, beverages, ice-cream, confections, preserves, cereals.

Orange

They are often used as “a lake” form – an insoluble aluminum complex.

Common Food Uses Cereals, baked goods, snack foods, ice-cream, beverages, dessert powders, confections

60

Tissue Stains • 

In the biological sciences these compounds are used as dyes to selectively stain different tissues or cell structures

• 

Biebrich Scarlet - Used with picric acid/aniline blue for staining collagen, recticulum, muscle, and plasma. Luna's method for erythrocytes & eosinophil granules. Guard's method for sex chromatin and nuclear chromatin. HO O3S

N N

N N SO3

61

pH Indicators • 

In the chemical sciences these are the acid-base indicators used for the various pH ranges:

• 

Remember the effects of pH on aromatic substituents

Methyl Orange

O3S

N N

Yellow, pH > 4.4

CH3 N CH3

O3S

H N N

CH3 N CH3

Red, pH < 3.2

62

Porphyrin Chromophore

Soret band

Q bands

Hemoglobin

deoxy

oxy

UV/Vis Analysis • 

•  •  • 

• 

A single band (e = 10 ~ 100) in 250 ~ 300 nm, no major absorption in 200 ~ 250 nm. n π*, C=O (ketone and aldehyde), C=N. N=N, -NO2 etc Two bands (e = 1,000 ~ 10,000) > 200 nm. π π*, aromatics Bands (e = 10,000 ~ 20,000) > 210 nm. π π*, conjugated C=C and C=O Compounds that are highly colored A long-chain conjugated system A polycyclic aromatic system pH-depended absorption Ionizable group attached to chromophore