UV-VISIBLE

SPECTROPHOTOMETRY

Objectives

To understand the process of absorption of light by molecules

To understand the relationships between Absorbance,
Transmittance, path length and analyte concentration (BeerLambert Law).
To be familiar with the quantitative applications of the Beer-Lambert
Law in the multi-component analysis of mixtures, and in standard
addition.
To be aware of the chemical and instrumental limitations of the
Beer-lambert Law.
To be aware of the major components of the instruments used for
measurement of absorbance.
To understand the experimental requirements for
spectrophotometric measurements.
To be familiar with typical applications of spectrophotometric
analysis.
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1. Introduction
Spectrophotometry - measurement of
light absorption to quantify
concentration of analyte
1.1 Light Absorption
Absorption of a photon by a molecule results in an excited state
Different wavelengths of light can be absorbed, which cause
different forms of excitation

1. Introduction
Electronic
excitation

Cosmic
rays

1020
-rays

1018
X-rays

Vibration

1016
UV

1014

Visible

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

Rotation

1012
Infrared

108
Microwave

Visible Spectrum

400

500

600

700

Radio

Bond breaking
and ionization

1.

Introduction

Bonding in organic molecules is based on overlap between and

atomic orbitals.
Electrons in and orbitals can be excited to the antibonding * and *
orbitals - involves absorption of different energies/wavelengths.

1.

Introduction

* and * transitions are

more likely than n * and n *
Stronger absorptions occur for
these transitions.
Wavelength and intensity of
absorption is affected by:

molecular geometry of

* (antibonding)
* (antibonding)

n (non-bonding)
(bonding)
(bonding)

molecule

types of substituent groups

nature of solvent (polarity)
The part of the molecule which
absorbs light is called the
chromophore.

Electronic energy levels of

polyatomic molecules

1. Introduction
1.2 Complementary Colours
When white light is absorbed by a chromophore, the eye detect
the colours that are not absorbed. This is called the
complementary colour to the colour absorbed.
Determination of concentration depends on detection of change
in colour intensity (absorption) at a particular wavelength.
of maximum
absorption

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

2. Colorimetric Analysis
2.1 Photometric measurement

(a) visual comparison using colour standards

Po

2. Colorimetric Analysis
(b) Colorimeter/Photometer

Filters used to select narrow wavelength

Detection with photosensing device

Filter
wheel

Po

Photodetector

2. Colorimetric Analysis
2.2

Spectrophotometric Analysis

Spectral bandwidth 1 nm, i.e very monochromatic light.

can operate in visible and UV ranges
Colorimetry and spectrophotometry provide sensitive methods of
analysis, i.e. ppm to ppb ranges.

Po

Monochromator

Photodetector
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3. Quantifying Light Absorption

b

Po
Reflected
beam

Pa

Pr
Absorbing solution
of concentration,c.

3.1 Incident Light

P0 = Pr + Pa + P
Pr 4% for air-glass interface;
I.e.use blank to zero P0 = Pa +

(1)

(2)

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3. Quantifying Light Absorption

3.2 Lamberts Law
For monochromatic light passing
though a transparent medium:
-dP

= - k P db
P=P

dP

P
P=Po

b=b

=k

db

b=0

P0
ln P = kb

Absorbance, A, defined as:

P0
A = log P
A = kb

3.3 Beers Law

The intensity of a monochromatic
light beam decreases with
concentration increase.

dP
= - k' dc
P
P

dP

P
Pa

= k' dc
0

P0
ln P = k'c or,
P0
log P = k"c

A = k"c
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3. Quantifying Light Absorption

3.4 Beer-Lambert Law

Combining these gives:

A=abc

Where:
a is the absorptivity, b is the path length,
length and c is the
concentration.
When c is in mol/L, and b is in cm,
A=bc
Where is the molar absorptivity.
absorptivity
Transmittance defined as:
T = P/Po
Hence:
A = log (1/T) = log(100/%T) = 2 - log(%T)
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4. Applications of the Beer-Lambert Law

4.1

Analysis of a single analyte

At fixed and b, is constant for a given chromophore.

Add chromogenic reagents to sample and standards, and measure
absorbance.
Assumes that the chemical matrix of standard is the same as the
sample.
A4
A3
Ax
A2
A1
A0
C0

Cx

Concentration

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4. Applications of the Beer-Lambert Law

4.2 Analysis of mixtures

Beer-Lambert Law applies to mixtures of of non-interacting components.

At 1 :
A1 = 1,M bcM + 1,N bcN,
at and 2:

A2 = 2,M bcM + 2,N bcN

1,M,

1,N, 2,M, 2,N are found from separate calibrations at 1 and 2. b is a

constant.
0.6
0.5
0.4
M
N
M+N

0.3
0.2

0.1

0.0
300

400

500

Wavelength (nm)

600

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4. Applications of the Beer-Lambert Law

Measure A1 and A2 at 1 and 2, and solve for cM and cN.

Accuracy decreases as the number of components increases.

Most accurate when differences between values are the greatest,

i.e choose appropriate values.

See Problem 5

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4. Applications of the Beer-Lambert Law

4.3

Standard Addition
-Multi Point Method

Used for samples with complex

matrix chemical interference if
calibration method applied.

Measure A of sample+ chromogenic

reagent.

Repeat with added increments of

standard to same vol. of sample.

Vs

0
Vol. Std added (mL)

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4. Applications of the Beer-Lambert Law

Before addition:

After addition:

A=

A =

b Vx Cx
Vt
b Vs Cs b Vx Cx
+
Vt
Vt

Sample Conc = Cx, Vol sample = Vx , Vol std added =Vs. Std Conc =
Cs, Total vol = Vt.

Plot A vs Cs, gives straight line: A = + Vs

Where = bCxVx/ Vt, and = bCs / Vt

Hence:
Cx = Cs / Vx
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4. Applications of the Beer-Lambert Law

4.4

Standard Addition-Two Point Method

See problem 4

Before addition:

A1 =

b Vx Cx
Vt

After addition:

A2 =

b Vs Cs b Vx Cx
+
Vt
Vt

and hence:

Cx =

A1 Cs Vs
(A2 - A1)Vx

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5. Limitations of the Beer-Lambert Law

5.1 Concentration effects
B-L law applies to dilute
solutions (negligible interaction
between solute ions).
Higher concentrations of
analyte (i.e. > 10-2M) or high
electrolyte concentrations,
molecular/ionic interactions
reduced light absorption at
some 's.
= f(n, refr. index of soln). If
changes in n large, deviation
from B-L observed.
Replace with n/(n2 + 2)2 in
B-L equation

Adherence to B-L law

Deviation from B-L law

(loss of sensitivity)

Concentration

20

5. Limitations of the Beer-Lambert Law

5.2 Instrumental deviations
Occur when polychromatic rather than monochromatic light is
used.
For a light beam consisting of ' and , B-L Law becomes:

A = log(P 0' + P0") - log(P0'10-'bc + P0"10-"bc)

Only when ' " is B-L law obeyed, i.e A = e'bc

' "

' "
Band A

' "

' < "

Band A

Band B

Band B

Wavelength

Concentration

21

6. Experimental Considerations
6.1 Wavelength selection
Choose where A is large to obtain best sensitivity.
Choose where dA/d = 0 or is small.

Absorbance




Wavelengt h

22

6. Experimental Considerations
6.2 Chromogenic reagents for colorimetric analysis

Should be stable and pure,

Should react rapidly with analyte to give a stable chromophore.

Should not absorb at of measurement, but.

Absorptivity, , should not be sensitive to minor pH, T, electrolyte

changes, etc.

Should be selective.

For UV measurements, often measure particular chromophore

directly, I.e. no reagent necessary (e.g. nitrate in GDR expt).
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7. Instrumentation
7.1

Principle

Light
Source

Wavelength
selection

Sample/
Blank

Signal
detection

Data/
Output

7.2 Photometer/Colorimeter
Source:
selector:

Generally visible light (Tungsten)

Filters, simple monochromator

Sample blank:

Single beam, no ref.

Detector:

Photocell or photodiode.

Output:

Microammeter/galvanometer/digital

Advantages / Disadvantages:
Lower cost
Light source fluctuations during measurement.
Light poorly monochromated.

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7. Instrumentation

Single beam spectrometer

25

7. Instrumentation

26

7. Instrumentation
7.3

UV-visible Spectrophotometer
Source:

UV-deuterium,visible-quartz halogen

selector:

Grating or Prism monochromator, scanning.

Sample/blank:Dual beam, can reference source fluctuations

Detector:

Phototube, photodiode, photomultiplier.

Output:

Digital, VDU,

Advantages:

More sensitive,
UV and visible spectra,
Scanning or diode array simultaneous spectral aquisition
better resolution (narrow spectral bandwidth)

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7. Instrumentation

Scanning, double beam UV-vis spectrometer

28

7. Instrumentation

Diode array spectrometer

29

7. Instrumentation
7.4 Probe photometers
To Detector
Optical filter

Optical fibre bundle

Mirror

30

7. Instrumentation
7.5 Optical Sensing Devices (Optrodes, Optodes)

Reflectance spectroscopy
Used for miniaturized in-situ measurement of glucose, pH
To Detector

Optical fibre bundle

31

8.Applications

Clinical,environmental, industrial and forensic

Screening of drugs
e.g. UV spectral identification and quantitation of amphetamines

Determination of cyanide
e.g. in Tylenol tablets

Use of UV-vis for detection after liquid, paper or thin-layer

chromatographic separation

Collect,
extract,
analyse
by UV-Vis
Sample

Std 2

Elution

Std 1

Pump

Flow-through
UV-vis spectrophotometer

Concentration
Time/ Distance

32

7. Instrumentation
7.6 Flow Injection Analysis

34

35

36

8. Applications
Rapid Screening

White powder is seized in a police

raid. Is it heroin?
UV spectrum of heroin shows max at
278 nm gives tentative identification.
Allows elimination of other materials
(e.g. starch, sugar, which are common
diluents used in heroin).
Allows thousands of other substances
to be eliminated.
Need to do confirmatory test, eg gas
chromatography, or mass spec.

37

8.Applications
Determination of
ethanol in expired
breath (The
Breathalyser)
2K2Cr2O7 + 3C2H5OH 8H2SO4
2Cr2(SO4)3 + 2K2SO4 +
3CH3COOH + 11 H2O

Measure K2Cr2O7 at 420

nm
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8.Applications
Screening (Fuel cell) breath tester
Absorbed methanol reacts giving small current.
Approx 3000 tests,

39