MOLECULAR ABSORPTION

SPECTROPHOTOMETER
IN UV-Vis WAVELENGTH

Prepared by : MSc. Huy Nguyen Dang

May 3, 2017 SCIENCE OF NATURE 1

 The Electromagnetic Spectrum

Gamma Rays X Rays UV IR Radar TV Radio

nm cm meters
0.001 0.01 0.1 1 10 100 1000 0.001 0.01 0.1 1 10 1 10 100 1000

UVB UVA Near IR

200 300 400 500 600 700 800 900

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 The Electromagnetic Spectrum

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 Molecular spectroscopy is based on the

UV, Visible, Infrared and Near-infrared spectroscopy measures

absorbed radiation
Fluorescence spectroscopy measures absorbed and emitted
radiation
Polarimetry measures the rotation of radiation

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interaction of molecules with electromagnetic radiation
 UV-VIS Spectrometer

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 Relationship between light absorption and color

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 UV-VIS Spectrometer
Source

Monochrometer

Slit Detector
Sample
I
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 UV-VIS Spectrometer

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 What Is Measured
Transmitted light = I
Incident light = Io

Transmittance = I/Io

%T = Transmittance x100

Absorbance = log(1/Transmittance)

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 Absorbance Vs Transmittance
0.10

0.09

ABS
0.08

Coffee Sample
0.07

0.06

0.05
A
0.04

0.03

0.02

0.01

0.00
250 300 350 400 450 500 550 600 650 700 750 800 850
NM
100

98

96

94

%T 92

90
%T
88

86

84

82

80
250 300 350 400 450 500 550 600 650 700 750 800 850
NM
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 Beers Law
I
A( ) ( ) b c log 0
I
=molar extinction coefficient, M -1 cm -1
b = pathlength, cm
c = concentration, Moles per liter
I0 = incident light
I = transmitted light
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 Cuvette Based Absorbance

I0 1 cm

A log
I I0 I
100%T means 0 AU
10% T means 1 AU
1% T means 2 AU
0,1% T meand 3 AU

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 Cuvette Based Absorbance

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 SINGLE BEAM

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 DOUBLE BEAM

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 (a) Single beam, (b) Double-beam-in-space

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 (a) Double-beam-in-space (c ) Double-beam-in-time
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 MY QUANG PH UV-VIS

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 LIGHT SOURCE HALOGEN LAMP

Many types of halogen

lamps :
6 V / 10 W
12 V / 20 W
24 V / 50 W
Vertical Filament
Horizontal Filament
Lifetime : > 2000 hours

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 LIGHT SOURCE

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 STRUCTURE OF DEUTERIUM LAMP

Filament applied Voltage :

5 V to 10 V
Hight voltage applied between anode
and cathode :
120 VDC to 200 VDC
Normal current : 300 mA
Lifetime : 1000 hours to 1500 hours

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 DEUTERIUM SPECTRUM

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 DEUTERIUM SPECTRUM

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 DEUTERIUM LIFETIME

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 FILTER

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 GRATING MONOCHROMATOR

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 GRATING CONSTRUCTION

1200 Grooves / 1mm

Aluminum is primarily
used as the reflective
material for grating.

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 STRAY LIGHT

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 OPTICAL SYSTEM

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 OPTICAL SYSTEM

Lens

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 DETECTORS

Many kinds of Detectors : photodiod, photomultilier, photodiod array

Photomultipllier construction 11 dynodes.

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 DETECTORS

Photodiod array

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 CUVETTE

CUVETTE Usable range

Square cuvette / Flowcell 200 nm to 1100 nm

Quartz
Square cuvette / Flowcell 320 nm to 1100 nm
Optical Glass
Test tube 340 nm to 1100 nm
Optical Glass

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