1

Chapter 6

UV –VIS SPECTROMETER

Spectroscopy

UV-VIS Spectrophotometer

Application

Case Study

Spectroscopy

• The procedures that uses light to measure

chemical concentration

• Light is described as photon because of its

ability to carry energy, E, that is given by

E = hv

where : h is a Plank constant (6.626x10-34 J.s)

v is frequency of light in Hz

Spectroscopy

• Spectroscopic analytical method are based on

measuring the amount of radiation produced or

absorbed by molecular or atomic species of

interest

• We can classify spectroscopic methods

according to the region of the electromagnetic

spectrum involved in the measurement.

• The region that have include such as X-ray,

Ultra violet, Visible, Infrared(IR) and etc

The Electromagnetic Spectrum

10 1001 10001010.10.010.00110001001010.10.010.001

Gamma Rays X Rays UV IR Radar TV Radio

nm cm meters

200 300 400 500 600 700 800 900

UVB UVA Near IR

visible

2

Electromagnetic Spectrum

• Regions which represent molecularprocesses that occur when light in eachregion is absorbed.

• Electromagnetic radiation in the UV-VISportion of the spectrum ranges inwavelength from approx. 200 to 700 nm.The UV range runs from 200 to 350 nmand the VIS range from 350 to 700 nm.

Absorption

• The process by which energy is

transferred from an electronic wave to an

atom or molecule and causes it to move to

an excited state.

• Absorption can only occur when an atom

or molecule absorbs a photon of light that

has an energy which exactly corresponds

to the difference between two energy

levels, that is it must be quantized.

• If an atom or molecule is subjected to

electromagnetic radiation of different

wavelengths (energies) it will only absorb

photons at those wavelengths which

correspond to exact differences between

two different energy levels within the

material.

Chromophore

• Absorption of UV VIS radiation results from the excitation of electrons from ground to excited states.

• Nuclei inside the molecules play an important role in:

– Determining wavelength of radiation absorbed

– Determine the strength with which the electrons are bound and thus influence the energy spacing between ground state and excited state.

Chromophore

• Hence the energy of transition and the

wavelength of radiation absorbed are

properties of a group of atoms rather than

the electron themselves called

chromophores.

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Beer’s Lambert Law

Beer’s Lambert law states the relationship between the

absorbance of a solution and the concentration of the

absorbing species.

A = a b c (1)

Where a = Proportional equation; absorptivity (Lg-1cm-1), b =

optical pathlength (cm) and c = Concentration (g L-1).

A = εεεε b c (2)

Where A= absorbance, εεεε = molar absorptivity (L mol-1cm-1), b

= optical pathlength (cm) and c = Concentration (mol L-1).

Beer law also known as absorption law or beer-Lambert law

• This Law tell us quantitatively how the amount ofattenuation depends on the concentration of theabsorbing molecules and the pathlength overwhich absorption occur.

• Absorption may be presented as:

Transmittance ( T = I / Io ) or Absorbance ( A = log Io / I )

b

Io I

Absorbing solution of concentration c

Note: Transmittanceransmittance isis oftenoften

expressedexpressed asas percentagepercentage andand

calledcalled percentpercent transmittancetransmittance

((%%T=T= TT xx 100100))

Absorbance VS Transmittance

The absorbance is a logaritma function of transmittance,

A = - log T

Note:

Since transmittance is often expressed aspercentage and called percent transmittance(%T= T x 100 %), A also can calculate as,

A = 2 – log %T

Where 2 = log 100

Note:

1. Absorbance is zero when transmittance is

100%, ie. No light absorbed by the sample

I = Io

T = 1.0 or 100 % and A = 0

2. Absorbance is infinity when transmittance is 0%,

ie. all light absorbed by the sample

I0 I

b=1 cm

Transmitted light = I

Incident light = Io

Figure 3 :

The relationship between absorbance and transmittance

If all the light passes through a solution

without any absorption, then absorbance is

zero, and percent transmittance is 100%. If

all the light is absorbed, then percent

transmittance is zero, and absorption is

infinite.

Absorption

• The process by which energy is

transferred from an electronic wave to an

atom or molecule and causes it to move to

an excited state.

• Absorption can only occur when an atom

or molecule absorbs a photon of light that

has an energy which exactly corresponds

to the difference between two energy

levels, that is it must be quantized.

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• If an atom or molecule is subjected to

electromagnetic radiation of different

wavelengths (energies) it will only absorb

photons at those wavelengths which

correspond to exact differences between

two different energy levels within the

material.

absorption spectroscopy

The amount of light absorbed as a function of

wavelength is measured, which give qualitative

and quantitative information about sample

The wavelength of maximum absorbance is a

characteristic value, designated as λ max

ABS

%T

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

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

NM

A

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

80

82

84

86

88

90

92

94

96

98

100

NM

%T

Coffee Sample

• Different compounds may have very differentabsorption maximum ( λ max) and absorbances.

• Intensely absorbing compounds must beexamined in dilute solution, so that significantlight energy is received by the detector, and thisrequires the use of completely transparent(non-absorbing) solvents.

• The most commonly used solvents are water,ethanol, hexane and cyclohexane. Solventshaving double or triple bonds or heavy atoms(e.g. S, Br & I) are generally avoided.

• Most spectrophotometers display

absorbance on the vertical axis, and the

commonly observed range is from 0

(100% transmittance) to 2 (1%

transmittance).

• Why do we commonly obtain and utilize

absorbance values rather than transmittance

values to quantitative compounds by UV –

Vis Spectroscopy ?

Path length /

cm 0 0.2 0.4 0.6 0.8 1.0

%T 100 50 25 12.5 6.25 3.125

Absorbance 0 0.3 0.6 0.9 1.2 1.5

Standard calibration graph

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• A = εεεε bc tells us that absorbance depends on

the total quantity of the absorbing compound in

the light path through the cuvette.

• If we plot absorbance against concentration, we

get a straight line passing through the origin

(0,0).

• The linear relationship between concentration

and absorbance is both simple and

straightforward,

• which is why we prefer to express the Beer-

Lambert law using absorbance as a measure of

the absorption rather than %T.

�ote that the

Law is not

obeyed at high

concentrations.

Some factors causing deviation from Beer-

Lambert Law :

�Gravimetric errors – eg. mood

�Incomplete spectral resolution – due to wrong slit

width selection

�Turbidity – must filter the sample first to avoid

cloudy

�Aggregation at high concentration – the particles

stick together

�Contamination of cuvettes – must clean the

cuvettes immediately after using

Using the Beer Law

Beer law expressed in equation 1 and 2 , can be used in several different way:

1. Calculate molar absortivity of species if theirconcentration are known

2. Can use to measure value of absorbance to obtainconcentration if absorptivity and pathlength are known.

3. More often , we use a series of standards solution of theanalytes to concentration to construct a calibrationcurve, or working curve , of A versus c (see figure 3).Then it can be used to determine the concentration ofunknown.

Example 1A 7.50 x 10 -5 M solution of potassium permanganatehas a transmittance of 36.4% when measured in a 1.05cm cell at wavelength of 525 nm. Calculate

(a) The absorbance of this solution

(b) the molar absortivity of KMnO4.

Answer:

A = - log T = - log 0.364= 0.439

Using equation 2,

ε = A / bc = 0.4389 / 1.05 cm x 7.50 x 10-5 mol L-1

ε = 5.57 x 103 Lmol-1cm-1

Example 2

At 580nm,The Wavelength of its maximum absorption,

the complex Fe(SCN)2+ has a molar absorptivity of

7.00 x 103 Lmol-1.Calculate the absorbance of a 2.50 x

10-5 M solution of the complex at 580 nm in 1.00-cm

cell

Answer:

A = εεεε b c

= (7.00 x 103 Lmol-1)(1.00-cm) (2.50 x 10-5 molL-1)

= 1.75 x 10-7

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Example 3 UV – VIS pectrophotometer Animation

Instrumentation and Basic

Components

Schematic diagram of a double-beam UV-

VIS spectrophotometer

Double Beam Instrument

Perkin Elmer Lambda 25

Lambda 25/35/45

Lambda 25 Optic diagram

Sources (UV and Visible)

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Types of Spectroscopy Source

A. Continuum Sources

Provides a broad distribution of wavelengthswithin a particular spectral range. Thisdistribution is known as a spectral continuum

B. Line sources

which emit a limited number of spectral lines,each of which spans a very limited wavelengthrange.

Sources of UV radiation

1. Deuterium ( and also Hydrogen ) lamps

Electrical excitation of deuterium or hydrogen

at low pressure produces a continuous UV

spectrum.

The mechanism involves formation of an

excited molecular species, which breaks up to

give atomic species and an ultraviolet photon.

Sources of UV radiation

� Both deuterium and hydrogen lamps emit

radiation in the range 160 – 375 nm.

Quartz windows must be used in these

lamps, and quartz cuvettes must be

used, because glass absorbs radiation of

wavelengths less than 350 nm.

Sources of visible radiation

2. Tungsten filament lamp

� commonly employed as a source of visible light.

� This type of lamp is used in the wavelength range of 350 – 2500 nm.

� The energy emitted is proportional to the fourth power of operating voltage. For stable energy output, the voltage to the lamp must be very stable. Electronic voltage regulators or constant-voltage transformers used to ensure this stability.

Sources of UV- ViS radiation

3. Tungsten/halogen lamps

Very efficient, and their output extends well

into the ultra-violet. They are used in many

modern spectrophotometers.

The lifetime of a tungsten/halogen lamp is

approximately double that of an ordinary

tungsten filament lamp.

2. Wavelength selector

(monochromator)

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Wavelength Selector

(monochromator)

� An entrance slit

� A collimating lens

� A dispersing device (usually a

prism or a grating)

� A focusing lens

� An exit slit

The aim of a monochromator is to isolate narrow

band of wavelengths centered on selected λ . We

want to have high intensity at the selected λ and

minimum light of other λ ‘s. All monochromators

contain the following component parts:

Polychromatic radiation (radiation of more

than one wavelength) enters the

monochromator through the entrance slit.

The beam is collimated, and then strikes

the dispersing element at an angle. The

beam is split into its component

wavelengths by the grating or prism. By

moving the dispersing element or the exit

slit, radiation of only a particular

wavelength leaves the monochromator

through the exit slit.

Czerney-Turner Grating Monochromator

3. Cuvettes

(sample containers)

sample containers

Sample Container, usually called cells or Cuvettes, must

have windows that are transparent in spectral region of

interest.

Types of material normally used are:

1. Quartz or fused silica required for the UV region (wavelength less than 350 nm) and may used in the visibleregion and out to about 3000 nm in the IR region.

2. Silicate Glass ordinary used for the region of 375 to2000nm because of its low cost compare quartz

3. Plastic cells also used in visible region Sample

The most common cell pathlength for studies in the UV – VIS

region is 1 cm.

The containers for the sample and reference

solution must be transparent to the radiation

which will pass through them. Quartz or fused

silica cuvettes are required for spectroscopy in

the UV region. These cells are also transparent

in the visible region. Silicate glasses can be

used for the manufacture of cuvettes for use

between 350 and 2000 nm.

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Different Types of Cuvettes Cell and Cell Holders

• standard • cylindrical• Long path

length

Handling of Cell

Clean and Dry Immediately after used

• Avoid scratches on polished cell windows

Cleaning of Cell

• Use the solvent in which the substance was dissolved

• After cleaning rinse with distilled water

• Never use hydrofluoric acid for cleaning – will etch the surface

• Never use ultrasonic cleaning – may cause cavitation

Detector

� Silicon photodiode array

� Vacuum phototube

� Photomultiplier tube

Usually uses photo-electric devices. It converts the

radiant energy (hv) into an electrical signal.

Examples of detectors are:

Type of Instrument

a) Single -Beam UV – VIS Spectrophotometer

B) Double Beam UV – VIS Spectrophotometer

• In single-beam uv-vis absorption spectroscopy,obtaining a spectrum requires manuallymeasuring the transmittance (see the Beer –Lambert Law) of the sample and solvent at eachwavelength.

• The double-beam design greatly simplifies thisprocess by measuring the transmittance of thesample and solvent simultaneously. Thedetection electronics can then manipulate themeasurements to give the absorbance

Single Beam VS Double Beam

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• It is well suited for quantitative absorption

measurement at single – wavelength type.

Here, simplicity of the instrumentation, low

cost, and ease of maintenance offer

distinct advantages.

Advantage of Single beam instrument Advantage of Double beam instrument

• Double beam instrument offer the advantage

that they are compensate for all but the most the

most short term fluctuation in the radiant output

of the sources.

• They also compensate for wide variation of

source intensity with wavelength. Furthermore,

the Double Beam design is well suited for

continuous recording of absorption spectra

Single-Beam UV-Vis SpectrophotometerDual-Beam uv-vis Spectrophotometer

Applying UV – VIS Molecular

Absorption Methods

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Absorption measurement in the UV – VIS region of

the spectrum provide qualitative and quantitative

information about organic, inorganic and biochemical

molecules.

Molecules absorbing UV – VIS radiation

Molecules absorbing UV – VIS

radiation

1.Absorption by Organic Species

• Species with unsaturated bonds generally absorb in theUV.

• Unsaturated organic functional groups that absorb in theUV – VIS region are known as Chromophores. Table listcommon chromophores and the approximate wavelengthat which they are absorb (Table 1.0).

• For many non nonabsorbing analytes, reagents areavailable that react with organic functional group toproduced species that absorb in the UV or Visiblereagent. For example: carbonyl containing compoundreact with dinitrophenylhydrazine to form colored speciesthat absorb at 480 nm.

Table 1.0 : Absorption Characteristic of Some Common Organic Chromophores

C h r o m o p h o r e E x a m p le S o lv e n t λλλλ m a x , n m εεεε m a x

A l k e n e C 6 H 1 3 = C H 2 n - h e x a n e 1 7 7 1 3 ,0 0 0

C o n j u g a te

A l k e n e

C H 2 = C H C H = C H 2 n - h e p t a n e 2 1 7 2 1 ,0 0 0

A l k y n e C 5 H 1 1 ≡≡≡≡ C - C H 3 n - h e p t a n e 1 7 8 1 0 ,0 0 0

1 9 6 2 ,0 0 0

2 2 5 1 6 0

C a r b o n y l

O

C H 3 C C H 3

n - h e x a n e

1 8 6

1 ,0 0 0

2 8 0 1 6

O

C H 3C C H

n - h e x a n e

1 8 0

L a r g e

2 9 3 1 2

O

C H 3 C � H 2

W a t e r

2 1 4

6 0

C a r b o n y l C H 3C O O H E t h a n o l 2 0 4 4 1

A z o C H 3 = � C H 3 E t h a n o l 3 3 9 5

� itr o C H 3 = � O 2 I s o o c ta n e 2 8 0 2 2

� itr a t e C 2 H O � O 2 D io x a n e 2 7 0 1 2

� itr o s o C 9 H 9 � O E t h y l E t h e r 3 0 0 1 0 0

6 6 5 1 0 0

A r o m a t ic B e n z e n e n - H e x a n e 2 0 4 7 9 0 0

2 5 6 2 0 0

2. Absorption by Inorganic Species

• In, general the ions and complexes of elements

in the first two transition series absorb broad

band of visible region in at least one of their

oxidation states and are, as consequence

colored. Example : Ni2+, Cr2O72-, Cu2+, C02+.

• Many inorganic species absorb in the UV – VIS

region. Ions such as nitrate, nitrite, and

chromate show characteristic UV – VIS

absorption

3. Absorption by Biochemical Species

• Many species of biochemical importance

also exhibit strong absorption in the UV –

Vis region.

• For example, NADH, the reduced form of

the coenzyme nicotinamide adenine

dinultide ( NAD+ ), absorb at 340 nm

Analysis using UV – VIS Spectrophotometer

Qualitative Analysis Quantitative Analysis

Even though it may not provide the

unambiquous identification of an organic

compound

nevertheless useful for detecting the

presence of certain functional group that act

as Chromophores

Example:

•Group containing phenyl, conjugated

double bond and the nitro group.

•Compilation of UV – VIS absorption

spectra are available to compare with the

absorption spectrum of a pure unknown

compound.

•Usually, UV – VIS absorption spectroscopy

is only used for conformation in conjunction

with more useful qualitative technique, such

as NMR, IR and Mass spectrometry

One of the most powerful and widely

used tools for quantitative

analysis.

Important Characteristics :

• Wide applicability to organic,

inorganic and biochemical

system

• Typical sensitivities of 10-4 to 10-

5 M

• Moderate to high selectivity

• Good accuracy ( Typically ,

relative uncertainties of 1% to

3% encounters)

• Ease and Convenience data

acquisition

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General UV – VIS Methods

• Simple measurement of absorbance and

transmittance values – A, % T

• Scanning – % A Scan, % T Scan

• Concentration Measurement

• Kinetic ( time Drive ) – Single wavelength

1.Scanning Methods

– Obtain typical spectrum of sample (using full range scan, 190 – 1100 nm)

2.Concentration Measurement

– Used for quantitative analysis

3.Kinetic Analysis

– Time based method

– Graphical real time display of absorbance vs

time ( study course of a chemical or

biochemical reaction with time)

– Used for enzyme kinetic study

Industrial Application

Typical Industrial Application

� Chemical analysis – quality control, quality analysis

� Biochemical analysis – proteins, DNA, enzymes

� Water and environment analysis – EPA & DIN methods,

cation/anion

� Kinetics – enzymes kinetics, substrate kinetic

� Food analysis – food colors (dyes), toxins, additives

(BHA, BHT), contaminants (heavy metals)

� Pharmaceutical – drugs (opium, heroin, cocaine)

Determining the concentration of an

unknown solution

Most dilute solutions obey the Beer-Lambert law and it can

be used to determine the concentration of an unknown

solution. Examples include

�the amount of iron in a sample of blood,

�the percentage of copper in brass (by first converting it

into a copper (II) ion solution, or

�investigating the reaction kinetics of a reaction involving

colored species.

The method is essentially the same in each case and is

illustrated by the following example.

A student was asked to plan an experiment to determine

the concentration of Cu2+ in an unknown solution. She was

provided with a separate solution of 1.00 mol dm-3 copper

(II) sulphate.

Using a spectrometer the student measured the

absorbance of a diluted sample of the copper (II) sulphate

solution at different wavelengths to obtain the value of 720

nm for λmax.

She then carefully diluted the 1.00 mol dm-3 solution to

give separate solutions with a range of known

concentrations.

The absorbance at 720 nm for each of these diluted

solutions was then measured.

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225.0 250 300 350 400 450 500.0

0.01

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.50

nm

A

225.0 250 300 350 400 450 500.0

0.01

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.50

nm

A

Concentration of CuSO4

(aq) / mol dm-3

Absorbance at 720 nm

0.100 0.362

0.150 0.498

0.200 0.798

0.250 0.901

0.300 1.002

0.500 1.751

The student used these results to plot a calibration curve using the line of

best fit. The absorbance of the unknown solution at 720 nm was then

measured.

0.00 0.5 1.0 1.5 2.0 2.5 3.00

0.00

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.00

mg/ml

A

∇

1

∇

2∇

3

Quantitatative analysis

Calculate the Relationship Between Absorbance and Concentration

Y = mx+C

225.0 250 300 350 400 450 500.0

0.01

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.50

nm

A

Scans of Potassium Dichromate solutions of different

Concentrations

Case Study

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Transmission spectra of two different suntan lotion showing

different levels of UVA and UVB light protection.

UVA=320-400nm(suntan) UVB=290-320nm(damage skin)

Transmittance Measurements

What to Look for When Shopping for a Sunglasses?

The standards for sunglasses place heavily in blocking UVB

and UVA rays. UVC are blocked by our earth's atmosphere

and does not reach the earth. According to the standards, to

effectively protect your eye's from harmful UV rays it must

block out at least 70% of UVB and 60% of UVA.

However, we suggest that to best protect your eyes look for

sunglasses that blocks out 98% of both UVB and UVA.

All our sunglasses we provide are made with UV400 nm

lenses. Which means it blocks out 100% of both UVB and

UVA rays. It exceeds industry standards and suggested

protection.

UVC

UVC is a type of magnetic waveform. It absorbs by the

earth's atmosphere below 280mm. 'The "C" is considered

a part of the UV family and has germicidal affects. When

around the 260-nanometer frequency, it is known to have

the highest affects. UVC is not harmful as compare to

UVA and UVB. However, with long and sustained

exposure it can make your skin read and your eyes feel

like there is sand in them. With proper protection from our

sunglasses, it can effectively block this type UV wave.

UVB causes cancer and burning of the eye. It can be

most damaging between 280 to 315 nanometer. It has a

shorter wavelength; therefore, it is known to cause

greater damage to the skin and eyes.

The medical community suggests any person tanning

indoor or outdoor to wear protective sunglasses. With

long exposure to UVB and UVA, it is known to cause skin

cancer and chronic eye diseases, such as cataract which

is clouding or hazy of the eye's lens.

UVB

UVA

UVA is a longer waveform than UVB. It

is the closest of the three waveforms to

visible light.

Most people get sunburns because of

UVA because of its longer waveform it

causes excitement in your skins cells

within the epithelial melanocytes.

UVA can also cause cataracts and

Pterygium within 300 to 300 nanometer.

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