Transcript Document

Spectroscopic ANALYSIS

Part 5 – Spectroscopic Analysis using UV-Visible Absorption

Chulalongkorn University, Bangkok, Thailand January 2012 Dr Ron Beckett Water Studies Centre & School of Chemistry Monash University, Melbourne, Australia Email: [email protected]

Water Studies Centre

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UV-Visible Absorption Spectroscopy Absorption of UV and visible light by a molecule causes electronic excitation

Bond breaking and ionization Electronic excitation Vibration Rotation 10 20

-rays 10 18 X-rays 400 500 10 16 UV 10 14 Infrared 10 12 10 8 Microwave Visible Spectrum 600 700

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UV-Visible spectral peaks result from electronic-vibrational transitions Case (b) in the diagram is most common which gives the typical symmetric peak shape 3

Molecular Orbitals • Bonding in organic molecules is based on overlap between

s

and

p

atomic orbitals .

• This can give rise to bonding orbitals, nonbonding

antibonding

s * and p

n

* s and p molecular molecular orbitals molecular orbitals Two

p

atomic orbitals overlapping to give a s and a s * molecular orbital 4

Molecular Orbitals

Two

p

atomic orbitals overlapping to give a bonding p molecular orbital and a nonbonding p * molecular orbital

A B

p *

A

+

B

p x p x

A B

5 p

Molecular Orbitals and Electronic Jumps s * (antibonding) p * (antibonding)

n

s * n p * n (non-bonding) p p * p (bonding) s s * s (bonding) Electronic energy levels of polyatomic molecules 6

Peak Position and the Type of Electronic Jump Conjugated p bonds 7

Peak Position for Molecules containing Double and Triple Bonds 8

Effect of Conjugation on Peak Position The greater the number of conjugated double bonds the lower the energy jump and higher the wavelength of the UV-visible peak p p * 9

Effect of Conjugation on Peak Position Highly conjugated molecules may be coloured if the absorption peak moves into the visible region 10

Question Time !

Fanta

has

red

and

green

colours !

Will

red

light pass through each of these solutions or will it be absorbed ?

(a) (b) (c) (d)

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Question Time !

Fanta

has

red

and

green

colours !

Will

green

light pass through each of these solutions or will it be absorbed ?

(a) (b) (c) (d)

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Complementary Colours

When white light is absorbed by a chromophore, the eye detects the colours that are

not

absorbed. This is called the complementary colour to the colour absorbed.

V I B G Y O R

Colour Absorbed Colour Observed  of  maximum absorption 380-440 440-500 500-580 580-680 680-780 violet-blue blue green green yellow orange-red purple green yellow orange-red violet-blue blue green green 13

Colorimetric Analysis

Used for determination of the concentration of analytes in solution when: 1. The analyte is a coloured compound 2. The analyte produces a coloured species when a suitable reagent is added 14

Colorimetric Analysis

Determination of concentration depends on detection of change in colour intensity (absorption) at a particular wavelength.

Photometric measurement (a) visual comparison using colour standards

P o P

Eye 15

Colorimetric Analysis

(b) Colorimeter/Photometer • Filters used to select a wavelength range • Detection with photosensing device

Filter wheel P o P Photodetector

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Spectrophotometric Analysis

(c) Spectrophotometer – Spectral bandwidth ≤ 1 nm, i.e very monochromatic light.

– can operate in both the visible and UV ranges – Colorimetry and spectrophotometry provide

sensitive

methods of analysis, i.e. ppm to ppb ranges.

P o P Monochromator

Prism or Grating

Photodetector

Phototube, photomultiplier 17 or photodiode

Single Beam Colorimeter

Single beam spectrometer 18

Quantifying Light Absorption

b

Incident beam

P I

Reflected

P r

beam P a(solute) P a(solvent)

Absorbing solution of concentration,c.

Transmitted beam

P

Incident Light Intensity (

P I

) (sometimes

I i

is used)

P I = P r + P a(solvent) + P a(solute) + P P a(solvent) & P a(solute) are absorbed light intensities P r

≈ 4% for air-glass interface 19

Quantifying Light Absorption Intensity lost due to reflection and solvent absorption are removed by measuring the transmitted intensity of a blank containing only solvent

b

Incident beam

P I

Reflected

P r

beam P a(solvent)

Absorbing solvent

Transmitted beam

P 0

Transmitted Light (

P 0

)

P I = P r + P a(solvent) + P 0 P 0 = P I - P r - P a(solvent)

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Quantifying Light Absorption

Absorbance is defined as P

A = log

P 0

Transmittance

defined as

T = P P 0

Thus A = log (1/T) = log(100/%T) 21

Relationship between Absorbance and Concentration

Beer-Lambert Law

A =

e

l c

Where: •

l

is the

path length in cm

• •

c

is the

concentration

in mol/L e is the

molar absorptivity

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Applications of the Beer-Lambert Law Analysis of a single analyte 1. Measure absorbance of a series of standard solutions 2. Plot a standard curve (should be a straight line ?) 3. Measure absorbance of unknown samples 4. Use standard curve to measure concentrations Assumptions – At fixed  and

l

, e constant for a given is solute – the chemical matrix of the standards is the same as the sample.

A 4 A 3 A x A 2 A 1 A 0 C 0 C 1

A =

e

l c

C 2 C C x 3 C 4

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Concentration

Applications of the Beer-Lambert Law Standard Addition Method Used for samples with complex matrix & chemical interferences.

1. Measure A of sample 2. Repeat with known additions of standard to the sample.

Sample plus standard additions Sample

C Add 0

Sample Concentration of Standard added (mL) Concentration 24

Limitations of the Beer-Lambert Law Concentration effects – B-L law applies to dilute solutions (negligible interaction – between solute ions).

Higher concentrations of analyte (i.e. > 10 -2 M) or high electrolyte concentrations, may produce molecular/ionic interactions which result in reduced light absorption at some wavelengths.

Adherence to B-L law Deviation from B-L law (loss of sensitivity) Concentration

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Experimental Considerations Wavelength selection • Choose  where A is large to obtain best sensitivity.

• Choose  where dA/d  = 0 or is small.

1 2 2 4 3

3

Wavelength 26

Experimental Considerations Choice of reagents for colorimetric analysis – Should be stable and pure – Should not absorb at  of measurement – Should react rapidly with analyte to give a stable coloured compound (chromophore).

– Absorptivity, e, should

not

be sensitive to minor changes in pH, Temp., electrolyte changes, etc.

– Should be selective for the analyte of interest.

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