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Spectroscopic ANALYSISPart 5 – Spectroscopic Analysis

using UV-Visible Absorption

Chulalongkorn University, Bangkok, Thailand January 2012

Dr Ron Beckett

Water Studies Centre & School of ChemistryMonash University, Melbourne, Australia

Email: Ron.Beckett@monash.eduWaterStudiesCentre

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UV-Visible Absorption Spectroscopy

1020 1018 1016 1014 1012 108

Cosm

ic

rays

-rays X-rays UV Vis

ible

Infrared Microwave

Electronic excitation

Bond breaking and ionization Vibration Rotation

Visible Spectrum

400 500 600 700

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

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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

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Molecular Orbitals

• Bonding in organic molecules is based on overlap between s and p atomic orbitals.

• This can give rise to bonding and molecular orbitals, nonbonding n molecular orbitals antibonding * and * molecular orbitals

Two p atomic orbitals overlapping to give a and a* molecular orbital

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Molecular Orbitals

+

px px

*

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

A

A B

A

B

B

6Electronic energy levels of polyatomic molecules

* (antibonding)

* (antibonding)

n (non-bonding)

(bonding)

(bonding)

Molecular Orbitals and Electronic Jumps

*

*

n * n *

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Peak Position and the Type of Electronic Jump

Conjugated bonds

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Peak Position for Molecules containing Double and Triple Bonds

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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

*

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Effect of Conjugation on Peak Position

Highly conjugated molecules may be coloured if the absorption peak moves into the visible region

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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

ofmaximumabsorption

Colour Absorbed Colour Observed

380-440 violet-blue green-yellow

440-500 blue-green orange-red

500-580 green-yellow violet-blue

580-680 orange-red blue-green

680-780 purple green

When white light is absorbed by a chromophore, the eye detects the colours that are notnot absorbed. This is called the complementary colour to the colour absorbed.

V I B G Y O R

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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

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Colorimetric Analysis

Photometric measurement

(a) visual comparison using colour standards

P Po

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

Eye

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Colorimetric Analysis(b) Colorimeter/Photometer

• Filters used to select a wavelength range

• Detection with photosensing device

PPo

Filter

wheel

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

PPo

PhotodetectorMonochromator

Prism or Grating Phototube, photomultiplier or photodiode

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Single Beam Colorimeter•

Single beam spectrometerSingle beam spectrometer

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Quantifying Light Absorption

Incident Light Intensity (PI) (sometimes Ii is used)

PI = Pr + Pa(solvent) + Pa(solute) + P

Pa(solvent) & Pa(solute) are absorbed light intensities

Pr ≈ 4% for air-glass interface

PPI

b

Absorbing solution of concentration,c.

Reflected beam

PrPa(solvent)

Pa(solute)Incident beam Transmitted beam

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Quantifying Light Absorption

Transmitted Light (P0)

PI = Pr + Pa(solvent) + P0

P0 = PI - Pr - Pa(solvent)

P0PI

b

Absorbing solvent

Reflected beam

Pr

Pa(solvent)

Incident beam Transmitted beam

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

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Quantifying Light Absorption

A = log P0

P

AbsorbanceAbsorbance is defined as

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

TransmittanceTransmittance defined as

T = PP0

Thus

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Relationship between Absorbance and Concentration

Beer-Lambert Law

A = l c

Where:• l is the path length in cmpath length in cm• c is the concentration concentration in mol/L • is the molarmolar absorptivityabsorptivity

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Applications of the Beer-Lambert Law

Analysis of a single analyte1. 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, is constant for a given solute

– the chemical matrix of the standards is the same as the sample.

Ax

Cx

4

3

2

1

0 2 3

A

A

A

A

A0

C C 1 C C C 4

Concentration

A = l c

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Standard Addition Method

Used for samples with complex matrix &chemicalinterferences.

1. Measure A of sample

2. Repeat with known additions of standard to the sample.

Applications of the Beer-Lambert Law

Concentration of Standard added (mL)

0CAdd

Sample

Sample plus standard additions

Sample Concentration

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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 inreduced light absorption at somewavelengths.

Concentration

Deviation from B-L law (loss of sensitivity)

Adherence to B-L law

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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.A

bso

rban

ce

3

Wavelength

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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, should not be sensitive to minor

changes in pH, Temp., electrolyte changes, etc.

– Should be selective for the analyte of interest.