APPLICATION OF MOLECULAR ULTRAVIOLET-

VISIBLE ABSORPTION SPECTROSCOPY

Introduction and Background

• Involves absorption of ultraviolet or visible radiation for qualitative and quantitative purposes.

• Most common analytical technique in the analytical laboratory

• Absorption commonly occurs with many– Organic molecules– Metals– Metal-organic complexes

ABSORBING SPECIES

The absorption of ultraviolet or visible radiation by an atomic or molecule can be considered to be a two step process :

M + hƲ M* M* M + heat

There are three types of electronic transitions :1.π, σ, and n electrons2. d and f electrons3.charge-transfer electrons

Ene

rgy

*

*

n

*

*

n

*

n

*

Antibonding

Antibonding

Nonbonding

Bonding

Bonding

Absorption by Organic Compounds

Many common organic compounds absorb in the UV region

Absorption by Inorganic Species

Many free metals and inorganic metal complexes absorb in the visible region of the spectrum

Absorption by Charge Transfer Complexes

• Many inorganic and organic complexes form charge transfer complexes

• A charge transfer complex consists of an electron donor group bonded to an electron acceptor group

• Charge transfer complexes exhibit broad band absorption in the visible region of the EMR spectrum

• Useful analytically because of the large molar absorption

What is Light?

• Light is a form of energy• Light travels through space at

extremely high velocities – The speed of light (c) ~ 3 x 1010

cm/sec or 186,000 miles per second

• Light is classified as electromagnetic radiation (EMR)

Characteristics of Light

• Light behaves like a wave.– That is, it can be modeled or characterized

with wave like properties.

• Light also behaves like a particle.

• Today, we envision light as a self-contained packet of energy, a photon, which has both wave and particle like properties.

The Electromagnetic Spectrum

EMR Wave Characteristics• Wavelength (l) is the distance from one wave

crest to the next.• Amplitude is the vertical distance from the

midline of a wave to the peak or trough.• Frequency (v) is the number of waves that pass

through a particular point in 1 second (Hz = 1 cycle/s)

Wave Properties of Electromagnetic Radiation

• EMR has both electric (E) and magnetic (H) components that propagate at right angles to each other.

Particle Properties of EMR

• The energy of a photon depends on its frequency (v)

Ephoton = hv

h = Planck’s constant

h = 6.63 x 10-27 erg sec or 6.63 x 10-34 Js

V = Wave Number (cm-1)l = Wave LengthC = Velocity of Radiation (constant) = 3 x 1010

cm/sec. u = Frequency of Radiation (cycles/sec)

 

The energy of photon:

h (Planck's constant) = 6.62 x 10-27 (Ergsec)

V =C

E = h = hC

C

= C =

Electromagnetic Radiation

How Light Interacts with Matter.

• Atoms are the basic blocks of matter.

• They consist of heavy particles (called protons and neutrons) in the nucleus, surrounded by lighter particles called electrons

How Light Interacts with Matter.

• An electron will interact with a photon.• An electron that absorbs a photon will

gain energy.• An electron that loses energy must

emit a photon.• The total energy (electron plus photon)

remains constant during this process.

Molecular Absorption

• More complex than atomic absorption because many more potential transitions exist– Electronic energy levels– Vibrational energy levels– Rotational energy levels

• Emolecule = Eelectronic + Evibrational + Erotational

Eelectronic > Evibrational > Erotational

• Result - complex spectra

Emission of EMR We distinguish several types of

emission1. Atomic2. X-Ray3. Fluorescence

Involves moleculesResonance and non-resonance modes

4. Phosphorescence• Non-radiative relaxation• Similar to fluorescence only relaxation

times are slower than fluorescence• Involves metastable intermediates

Energy Level Diagrams of Excitation and Emission

Auxokrom : gugus jenuh yang bila terikat pada kromofor mengubah panjang gelombang dan intensitas serapan maksimum. Ciri auxokrom adalah heteroatom yang langsung terikat pada kromofor, missal : -OCH3, -Cl, -OH dan NH2.Pergeseran batokromik : Pergeseran serapan kearah panjang gelombang Yang lebih panjang disebabkan substitusi atau pengaruh pelarut (pergeseran merah).Pergeseran hipsokromik : Pergeseran serapan kearah panjang gelombang yang lebih pendek disebabkan substitusi atau pengaruh pelarut (pergeseran biru).Efek Hiperkromik, kenaikan dalam intensitas serapan.Efek hipokromik, penurunan dalam intensitas serapan.

A few metal chlorides, which fluoresce strongly in the visible wavelengths,are the basis for almost all the colors in modern fireworks.

Barium chloride produces green; strontium chloride produces red; copper chloride produces blue

Single Beam Instruments

Double-Beam Instruments• A double beam instrument is one in

which the light source can be passed (simultaneously) through both a reference and a sample cell

• Purpose and Approach1. Adjust light output of the instrument

to 100% transmission (0 % absorbance)

2. Allows correction of the sample absorbance signal for non-analyte absorbance

Double-Beam Instruments

Reference and Sample Cell OptionsReference and Sample Cell OptionsReference Reference Reference Reference Sample or Standard Sample or StandardCellCell Cell Cell Cell CellSolution (pure HSolution (pure H

22O) Solution (pure HO) Solution (pure H22O) Solution (pure HO) Solution (pure H

22O)O)

ReagentsReagents Reagents Reagents Analyte or SampleAnalyte or Sample

Signal Due toReagents Only(Can be used to estimatereagent blank)

Signal Due toAnalyte Only

Example of UV-Visible Instrument

QUALITATIVE ANALYSIS• SOLVENTS

Transparant

Polar solvents [water, alcohols, esters,

ketones]

water 190 nm cyclohexane 210 nm ethanol 210 nm benzene 280 nm n-hexane 195 nm diethyl ether 210 nm

acetone 330 nm 1,4-dioxane 220 nm

• DETECTION OF FUNCTIONAL GROUPS

Spectroscopy Terms Describing Absorption (Beer’s Law)

• Consider a beam of light with an (initial) radiant intensity Po

• The light passes through a solution of concentration (c)

• The thickness of the solution is “b” cm.

• The intensity of the light after passage through the solution (where absorption occurs) is P

P0hv P

b

Co

nce

ntr

atio

n (

c)

We Define

• Transmittance (T) = P/P0 (units = %)

• Absorbance (A) (units = none)– A = log (P0/P)

– A = -log (T) = log (1/T)

– A = abc (or εbc) <--- Beer’s Law• a = absorptivity (L/g cm)• b = path length (cm)• c = concentration (g/L)• ε = molar absorptivity (L/mol cm)

– Used when concentration is in molar units

Beer’s Law

Major Point: There is a linear relationship

between absorbance and concentration (but not absorbance and transmission)

A = abc = εbc = log (Po/P) = log (1/T)

P0 = 10,000 P = 5,000

-b-

Example

A = -log T = -log (0.5) = 0.3010

P0 = 10,000 P = 2,500

25.010000

2500

0

P

PT

--2b--

Example

A = -log T = -log (0.25) = 0.6021

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 1 2 3 4 5 6 7 8 9 10

Thickness, multiples of b

Ab

sorb

an

ce Absorption vs. Absorption vs.

TransmissionTransmission

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6 7 8 9 10

Thickness, multiples of b

Tra

nsm

itta

nce

A = abc

T = 10-abc

Limitations to Beer’s Law

• Real– At high concentrations charge distribution effects

occur causing electrostatic interactions between absorbing species

• Chemical– Analyte dissociates/associates or reacts with

solvent

• Instrumental– ε = f(λ); most light sources are polychromatic

not monochromatic (small effect)– Stray light – comes from reflected radiation in

the monochromator reaching the exit slit.

Instrumental Limitations - ε = f(λ)

• How/Why does ε vary with λ?

• Consider a wavelength scan for a molecular compound at two different wavelength bands

• In reality, a monochromator can not isolate a single wavelength, but rather a small wavelength band

Instrumental Limitations – Stray Light

• Result – non-linear absorption (Analyte vs. Conc.) as a function of analyte concentration– Similar to

polychromatic light limitations

QUANTITATIVE ANALYSIS

• SELECTION OF WAVELENGTH• VARIABLES THAT INFLUENCE

ABSORBANCE• CLEANING AND HANDLING OF CELLS• DETERMINATION OF THE RELATIONSHIP

BETWEEN ABSORBANCE AND CONCENTRATION

• STANDARD ADDITION METHOD• ANALYSIS OF MIXTURES OF ABSORBING

SPECIES

SPEKTROFOTOMETRI DERIVATIF

• Mengalihbentuk data spektrum• Diperoleh dengan cara memplotkan turunan

pertama atau turunan lebih tinggi absorban terhadap panjang gelombang :

A = f(λ)

dA/d λ = f ‘(λ)

d2A/d λ2 = f “(λ)

dst

zero crossing

Zero order spectrum

A Gaussian absorption band and its first to fourth order derivatives

223 nm 266 nm

246 nm Thiamine

Riboflavine

Mixture of thamine and riboflavine

Thiamine

Riboflavine

223 nm

246 nm 266 nm

Chromophoric Structure

Group Structure nm

Carbonyl > C = O 280

Azo -N = N- 262

Nitro -N=O 270

Thioketone -C =S 330

Nitrite -NO2 230

Conjugated Diene -C=C-C=C- 233

Conjugated Triene -C=C-C=C-C=C- 268

Conjugated Tetraene -C=C-C=C-C=C-C=C- 315

Benzene 261

VITAMINS

Vitamin A H3C CH3

CH3

CH3

CH3

CH2OH

- Carotene

CH3

CH3

CH3 CH3 CH3

CH3 CH3 CH3

H3 C

CH3

H3 C CH3

CH3

CH3 CH3

H3 C CH3

CH3

CH3 CH3

CH2OH

Oxidation

C H

O

Retainal

Retinol (Vitamin A)

- 2H

Food

KOH (Alcoholic) Saponification

3 hrs. at room temperature

Ether for Extraction

Extract (vit.A and carotenoids)

Total Carotenoids only at 440 nm

Vitamin A + Carotenes Carr-Price Reagent Measure at 620 nm

Vitamin A and - Carotene Determination

-CAROTENE STANDARD ABSORBANCES AT 440 AND 620 nm

A a

t 440 n

m

an

d 6

20

nm

at 440 nm

at 620 nm

A

A

Use absorbances at 440 nm and then convert this to absorbance at 620 nm and subtract from the absorbance at 620 nm to determine the absorbance at 620 due to Vitamin A.

62 0

Carotenoid Absorbance at 440nm Vitamin A Absorbance at 620 nm

Carotenoid (g/ml) Vitamin A g/cuvette (sample)

Abs

orba

nce

at 4

40nm

Abs

orba

nce

at

n

mx

x

x

x

x

xx

x

x

x

x

THIAMIN DETERMINATION THIOCHROME (Fluorescent)

N

K3Fe(CN)6Oxidation

N

S

N

NH3C

CH2 CH3

CH2CH2OH

N

S

N

NH3C

CH2

NH2

CH3

CH2CH2OH

pyrimidine thiazole

Excite thiochrome at 365 nm and measure the absorbance at 435 nm

PHOTOMETRIC TITRATIONSPhotometric measurements can be employed to advantage in locating the equivalence of a titration, provide the analyte, the reagent, or the titration product absorbs radiation.

A photometric titration curve is a plot of absorbancecorrected for volume changes, as a function of the volume of titrant. If conditions are chosen properly,the curve will consist of two straight line regions with differing slopes, one occuring at the outset of the titration and the other located well beyond the equivalence region. The EP is taken as the intersection of extrapolated linear portions.

A(λ1) = aI(λ1) cI + aII(λ1) c II ……………………(1)A(λ2) = aI(λ2)cI + aII(λ2) cII ……………………… (2)Dimana :•nilai A(λ1 ) dan A(λ2) diperoleh dari pengukuran sample pada masing2 panjang gelombang maksimum.•aI(λ1) diperoleh dari harga tangens dari kurva baku komponen I pada λ1•aII(λ1) diperoleh dari harga tangens dari kurva baku komponen II pada λ1•aI(λ2) diperoleh dari harga tangens dari kurva baku komponen I pada λ2•aII(λ2) diperoleh dari harga tangens dari kurva baku komponen II pada λ2•CI dan CII adalah konsentrasi komponen I dan komponen II yang tidak diketahui