lesson 8 uv-vis spectroscopy.pdf

7/24/2019 lesson 8 uv-vis spectroscopy.pdf

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What is Spectroscopy?

The study of molecular structure and

dynamics through the absorption,

emission and scattering of light.


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What is Light?

According toMaxwell, light is anelectromagnetic field

characterized by afrequency f, velocity v( c in vacuum), andwavelength . Lightobeys therelationship

= c / .

Its energy is E= h


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The Electromagnetic Spectrum

= c / lE = h


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X-ray:

core electronexcitation

UV:

valenceelectronic

excitation

IR:

molecularvibrations

Radio waves:

Nuclear spin states(in a magnetic field)


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Spectral Distribution of Radiant Energy

Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)


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Spectroscopic Techniques

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


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UV = 300 kcal/mol

10-5

10-5


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Molecules have quantized energy levels:

ex. electronic energy levels.

energy

hv

energy

}= hv

Q: Where do these quantized energy levels come from?

A: The electronic configurations associated with bonding.

Each electronic energy level(configuration) has

associated with it the many

vibrational energy levels we

examined with IR.


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2s 2s

s

s*

s

s*

p*

p

2p 2pn


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C C

s

s*

hv

s

s*

s s*

C C

H

H

H H

HH

lmax

= 135 nm (a high energy transition)

Absorptions having lmax< 200 nm are difficult to observe because

everything (including quartz glass and air) absorbs in this spectral

region.

Ethane


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C C

s

s*

hv

p

p*

s

s*

p

p*

p p*

Example: ethylene absorbs at longer wavelengths:lmax= 165 nm = 10,000

= hv=hc/l


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s

s*

hv

p

p*

n

s

s*

p

p*

n

C O

p*n

The n to pi* transition is at even lower wavelengths but is notas strong as pi to pi* transitions. It is said to be forbidden.

Example:

Acetone: ns* lmax= 188 nm ; = 1860

np* lmax= 279 nm ; = 15


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C C

C C

C O

C O

H

s s* 135 nm

p p*165 nm

n s* 183 nm weak

p p* 150 nm

n s* 188 nmn p* 279 nm weak

l

A

180 nm

279 nm

C O


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C C

HOMO

LUMO

Conjugated systems:

Preferred transition is between Highest Occupied Molecular Orbital

(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).

Note: Additional conjugation (double bonds) lowers the HOMO-

LUMO energy gap:

Example:

1,3 butadiene: lmax= 217 nm ; = 21,000

1,3,5-hexatriene lmax= 258 nm ; = 35,000


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Lycopene:

lmax= 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm

lmax(Actual) = 474.


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Electronic (UV) spectroscopy Light absorbedelectron excited to higher

molecular orbital Transitions occur from HOMO to LUMO

- Highest Occupied Molecular Orbital

- Lowest Unoccupied Molecular Orbital

E=h


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What functional groups are observed

in UV/vis spectrum?

UV/vis (200-600 nm)

Functional groups

KetoneEster

Amide

Conjugated D.B

Not observedC-C

C-H

C=C (isolated)

C-C

LUMO s

HOMO s

C=C

LUMO p

HOMO p

C=C-C=C

LUMO

HOMO

l=217 nm

Excitation

p4

p3

p2

p

1

l=165nm

l=125nm


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Chromophores

Chromophores- Part of molecule responsible forabsorption

Auxochromes- Groups that modify absorption ofneighboring chromophores- Often have lone pairs, e.g.OH, -OR,

-NR2, -halogen- Bathochromic shift: towards longerwavelength

- Hypsochromic shift: towards shorterwavelen th


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The wavelength and amount of light that a compound

absorbs depends on its molecular structure and theconcentration of the compound used.

T itt d


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Transmittance and

Concentration

The Bouguer-Lambert Law

PathlengthConst

eIIT

0/


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Transmittance and Path Length:

Beers Law

ionConcentratConsteIIT 0/

Concentration


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BEER LAMBERT LAW

Glass cell filled with

concentration of solution (C)

IILight0

As the cell thickness increases, the intensity of I

(transmitted intensity of light ) decreases.


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T- Transmittance

T = I0- original light intensity

I- transmitted light intensity

% Transmittance = 100 x

Absorbance (A) or optical density (OD) = Log

= Log

Log is proportional to C (concentration of solution)

and is also proportional to L (length of light path

through the solution).

I

I0

I

I0

I0

I

1

T

I

I0


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A CL = KCL by definition and it is called

the Beer Lambert Law.

A = KCL

K = Specific Extinction Coefficient ---- 1 g of

solute per liter of solution

A = ECL

E = Molar Extinction Coefficient ----

Extinction Coefficient of a solution containing

1g molecule of solute per 1 liter of solution


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L = Cm

C = Moles/Liter

A = KCL

A = No unit C = Gram/Liter L = Cm

A = ECL = ( LiterCm x Mole

) x MoleLiter

x Cm

K=

Liter

Cm Gram

A = KLC = (Liter

Cm x Gram

Gram

Literx Cm) x

UV VIS SPECTROMETERS ARE UBIQUITOUS


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P 8

BEGIN

HERE

UV-VIS SPECTROMETERS ARE UBIQUITOUS


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Characteristics of UV-Vis spectra of

Organic Molecules

Absorb mostly in UV unless highlyconjugated

Spectra are broad, usually to broad for

qualitative identification purposes Excellent for quantitative Beers Law-type

analyses

The most common detector for an HPLC


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Broad spectra

Overlapping vibrational and rotational

peaks Solvent effects


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P354


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A = 0.8

Conc: 4x10-5M

Path: 1 cm

= 0.8/4x10-5x1

= 2x104

= 20000

Isoprene


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P356

(see also 359)


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Conventional

Spectrophotometer

Schematic of a conventional single-beam spectrophotometer


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Light Sources

Hydrogen Gas Lamp

Mercury Lamp

Tungten lamp (350-2500 nm)

Deuterium (200-400 nm)

Xenon Arc lamps (200-1000 nm)


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Dispersion Devices

Non-linear dispersion

Temperature sensitive

Linear Dispersion

Different orders


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Cells

UV Spectrophotometer

Quartz (crystalline silica)

Visible Spectrophotometer

Glass

IR Spectrophotometer

NaCl


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Open-topped rectangular standard cell (a)

and apertured cell (b) for limited sample volume

Cell Types I


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Conventional

Spectrophotometer

Optical system of a double-beam spectrophotometer


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STEPS IN DEVELOPING

SPECTROPHOTOMETRIC

N LYTIC L METHOD

1. Run the sample spectrum

2. Obtain a monochromatic

wavelength for the

maximum absorption

wavelength.

3. Calculate the concentration

of your sample using Beer

Lambert Equation: A = KCLWavelength (nm)

Absorbance

0.0

2.0

200 250 300 350 400 450


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Slope of Standard Curve = A

C

1 2 3 4 5

1.0

0.5

Concentration (mg/ml)

Absorbance at 280 nm

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where

becomes non-linear.

SPECTROMETRIC ANALYSIS USING


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SPECTROMETRIC ANALYSIS USING

STANDARD CURVE

1 2 3 4

0.4

0.8

1.2

Absorbance at 540 nm

Concentration (g/l) glucose

Avoid very high or low absorbencies when drawing a

standard curve. The best results are obtained with 0.1 < A

< 1. Plot the Absorbance vs. Concentration to get astrai ht line


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Every instrument has a useful range for a

particular analyte.

Often, you must determine that rangeexperimentally.

This is done by making a dilution series of

the known solution.

These dilutions are used to make a

working curve.


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Make a dilution series of a known quantity

of analyte and measure the Absorbance.

Plot concentrations v. Absorbance.


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What concentration do you think the

unknown sample is?


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In this graph, values above A=1.0 are not linear. If we

use readings above A=1.0, graph isnt accurate.

C lib ti M th d


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Calibration MethodsStandard Addition

1.) Protocol to Determine the Quantity of an Unknown(i) Known quantities of an analyte are added to the unknown

-known and unknown are the same analyte

- increase in analytical signal is related to the total quantity of the analyte

-requires a linear response to analyte

(ii) Very useful for complex mixtures

- compensates for matrix effect change in analytical signal caused by

anything else than the analyte of interest.

(iii) Procedure:

(a) place known volume of unknown sample in multiple flasks


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C lib ti M th d


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XS

X

ff

i

I

I

XS

X

Calibration MethodsStandard Addition

1.) Protocol to Determine the Quantity of an Unknown(iii) Procedure:

(d) Measure an analytical response for each sample

- signal is directly proportional to analyte concentration

solutionfinalfromsignal

solutioninitialfromsignal

solutionfinalindardtansplusanalyteofionConcentrat

solutioninitialinanalyteofionConcentrat

Standard addition equation:

V

VSS

V

VXX Sif

oif

dardtansofvolumeaddedV,volumeinitialunknownV,VVV SoSo

Total volume (V):


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VIS-Transmission and Color

The human eye sees the complementary color to that which is

absorbed


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lesson 8 uv-vis spectroscopy.pdf