MOLECULAR ABSORPTION

SPECTROSCOPY :

THEORY,

INSTRUMENTATION &

APPLICATION

CHAPTER 2

COMPONENTS OF

INSTRUMENTS FOR

OPTICAL SPECTROSCOPY

General Design of Optical

Instruments

Absorption

Emission

Five Basic Optical Instrument Components

1) Source – A stable source of radiant energy at the desired wavelength (or range).

2) Sample Container – A transparent container used to hold the sample (cells, cuvettes, etc).

3) Wavelength Selector – A device that isolates a restricted region of the EM spectrum used for measurement (monochromators, prisms & filters).

4) Detector/Photoelectric Transducer – Converts the radiant energy into a useable signal (usually electrical).

5) Signal Processor & Readout – Amplifies or attenuates the transduced signal and sends it to a readout device as a meter, digital readout, chart recorder, computer, etc.

I. Sources of Radiation

• Generate a beam of radiation that is stable and has sufficient power.

A. Continuum Sources – emit radiation over a broad wavelength range and the intensity of the radiation changes slowly as a function of wavelength.

This type of source is commonly used in UV, visible and IR instruments.

- Deuterium lamp is the most common UV source.

- Tungsten lamp is the most common visible source.

- Glowing inert solids are common sources for IR instruments.

B. Line Sources – Emit a limited number lines or bands of radiation at specific wavelengths.

- Used in atomic absorption spectroscopy

- Usually provide radiation in the UV and visible region of the EM spectrum.

- Types of line source:

1) Hollow cathode lamps

2) Electrodeless discharge lamps

3) Lasers-Light – amplification by stimulated emission of radiation

II. Wavelength Selectors

• Wavelength selectors output a limited,

narrow, continuous group of wavelengths

called a band.

• Two types of wavelength selectors:

1) Filters

2) Monochromators

A. Filters

- Two types of filters:

1) Interference Filters

2) Absorption Filters

B. Monochromators

- Wavelength selector that can continuously

scan a broad range of wavelengths

- Used in most scanning spectrometers

including UV, visible, and IR instruments.

III. Radiation Transducer (Detectors)

• Early detectors in spectroscopic

instruments were the human eye,

photographic plates or films. Modern

instruments contain devices that convert

the radiation to an electrical signal.

• Two general types of radiation transducers:

a. Photon detectors

b. Thermal detectors

A. Photon Detectors

- Commonly useful in ultraviolet, visible, and near infrared instruments.

- Several types of photon detectors are available:

1. Vacuum phototubes

2. Photomultiplier tubes

3. Photovoltaic cells

4. Silicon photodiodes

5. Diode array transducers

6. Photoconductivity transducers

B. Thermal Detectors

- Used for infrared spectroscopy because photons in the IR region lack energy to cause photoemission of electrons.

- Three types of thermal detectors:

1. Thermocouples

2. Bolometers

3. Pyroelectric transducers

IV. Sample Container • Sample containers, usually called cells or cuvettes must have

windows that are transparent in the spectral region of interest.

• There are few types of cuvettes:

- quartz or fused silica

- silicate glass

- crystalline sodium chloride

Quartz or fused silica

- required for UV and may be used in visible region

Silicate glass

- Cheaper compared to quartz. Used in UV.

Crystalline sodium chloride

- Used in IR.

Spectrometer

- is an instrument that provides information about the intensity of radiation as a function of wavelength or frequency.

Spectrophotometer

- is a spectrometer equipped with one or more exit slits and photoelectric transducers that pemits the determination of the ratio of the radiant power of two beams as a function of wavelength as in absorption spectroscopy.

SUMMARY

Types of source, sample holder and detector

for various EM region

REGION SOURCE SAMPLE

HOLDER

DETECTOR

Ultraviolet Deuterium lamp Quartz/fused silica Phototube, PM

tube, diode array

Visible Tungsten lamp Glass/quartz Phototube, PM

tube, diode array

Infrared Nernst glower

(rare earth oxides

or silicon carbide

glowers)

Salt crystal e.g.

crystal sodium

chloride

Thermocouples,

bolometers

ULTRAVIOLET-VISIBLE

SPECTROSCOPY

• Absorption process in UV/VIS region in

terms of its electronic transitions

• Molecular species that absorb UV/VIS

radiation

• Important terminologies in UV/VIS

spectroscopy

In this lecture, you will learn:

INSTRUMENTATION

Important components in a UV-Vis spectrophotometer

Source

lamp

Sample

holder selector Detector

Signal

processor

& readout

1 2 3 4 5

UV region:

-Deuterium lamp;

H2 discharge tube

Visible region:

- Tungsten lamp

Glass/quartz

Prism/monochromator

Phototube,

PM tube, diode

array

Quartz/fused silica

Prism/monochromator

Phototube,

PM tube, diode

array

Instrumentation

• UV-Visible instrument

1. Single beam

2. Double beam

Single beam instrument

• Single beam instrument

- One radiation source

- Filter/monochromator ( selector)

- Cells

- Detector

- Readout device

Single beam instrument

• Disadvantages:

– Two separate readings has to be made on the

light. This result in some error because the

fluctuations in the intensity of the light do

occur in the line voltage, the power source

and in the light bulb btw measurements.

– Changing of wavelength is accompanied by a

change in light intensity. Thus spectral

scanning is not possible.

Double beam instrument

Double-beam instrument with beams separated in space

• Double-beam instrument

Advantages:

1. Compensate for all but most short-term

fluctuations in the radiant output of the

source as well as for drift in the

transducer and amplifier.

2. Compensate for wide variations in

source intensity with .

3. Continuous recording of transmittance

or absorbance spectra.

MOLECULAR SPECIES THAT

ABSORB UV/VISIBLE RADIATION

INORGANIC

SPECIES

ORGANIC

COMPOUNDS

CHARGE

TRANSFER

Definitions: • Organic compounds

– Chemical compound whose molecule contain carbon

– E.g. C6H6, C3H4

• Inorganic species – Chemical compound that does not contain carbon.

– E.g. transition metal, lanthanide and actinide elements.

– Cr, Co, Ni, etc

• Charge transfer – A complex where one species is an electron donor and

the other is an electron acceptor.

– E.g. iron (III) thiocyanate complex

PERIODIC TABLE OF

ELEMENTS

• In UV/VIS spectroscopy, the transitions

which result in the absorption of EM

radiation in this region are transitions

between electronic energy levels.

ULTRAVIOLET-VISIBLE

SPECTROSCOPY

• In molecules, not only have electronic

level but also consists of vibrational and

rotational sub-levels.

• This result in band spectra.

Molecular absorption

Types of transitions

• 3 types of electronic transitions

- , and n electrons

- d and f electrons

- charge transfer electrons

H H O + + O H H O H H or

single covalent bonds (σ)

O C O or O C O

double bonds ()

N N N N

triple bond ()

or

What is σ, and n electrons?

lone pairs(n)

Sigma () electron

Electrons involved in single bonds such as

those between carbon and hydrogen in alkanes.

These bonds are called sigma () bonds.

The amount of energy required to excite

electrons in bond is more than UV photons of

wavelength. For this reason, alkanes and other

saturated compounds (compounds with only

single bonds) do not absorb UV radiation and

therefore frequently very useful as transparent

solvents for the study of other molecules. For

example, hexane, C6H14.

• Electrons involved in double and triple

bonds (unsaturated).

• These bonds involve a pi () bond.

• For exampel: alkenes, alkynes,conjugated

olefins and aromatic compounds.

• Electrons in bonds are excited relatively

easily; these compounds commonly

absorb in the UV or visible region.

Pi () electron

• Examples of organic molecules containing

bonds.

CH2CH3

C

C

C

C

C

C

H

H

H

H

H

H

C HCCH3

ethylbenzene benzene

propyne

C

H

C

C

H

HC

HH

H

1,3-butadiene

• Electrons that are not involved in

bonding between atoms are called n

electrons.

• Organic compounds containing nitrogen,

oxygen, sulfur or halogens frequently

contain electrons that re nonbonding.

• Compounds that contain n electrons

absorb UV/VIS radiation.

n electron

• Examples of organic molecules with non-

bonding electrons.

NH2

C

O

R

C C

H

HBr

H3C

:

.. :

..

.. : aminobenzene Carbonyl compound

2-bromopropene If R = H aldehyde

If R = CnHn ketone

• UV/Vis absorption by organic compounds

requires that the energy absorbed

corresponds to a jump from occupied

orbital to an unoccupied orbital of greater

energy.

• Generally, the most probable transition is

from the highest occupied molecular

orbital (HOMO) to the lowest unoccupied

molecular orbital (LUMO).

ABSORPTION BY ORGANIC COMPOUNDS

Electronic energy levels diagram

Antibonding

Antibonding

Nonbonding

Bonding

Bonding

Unoccupied levels

Occupied levels

n

*

*

Energ

y

*

*

n

*

n

*

Electronic transitions

*

*

n *

n *

In alkanes

In alkenes, carbonyl compounds, alkynes, azo

compounds

In oxygen, nitrogen, sulfur and halogen

compounds

In carbonyl compounds

Increasing

energy

* transitions

• The energy required to induce a * transition is large (see the arrow in energy level diagram).

• Never observed in the ordinarily accessible ultraviolet region.

• This type of absorption corresponds to breaking of C-C, C-O, C-H, C-X, ….bonds

Electronic transitions

- Saturated compounds containing atoms with unshared

electron pairs (non-bonding electrons).

- Compounds containing O, S, N and halogens can

absorb via this type of transition.

- Absorption are typically in the range, 150 - 250 nm

region and are not very intense.

- range: 100 – 3000 cm-1mol-1

- Absorption maxima tend to shift to shorter in polar

solvents.

e.g. H2O, CH3CH2OH

n * transitions

Some examples of absorption due to

n * transitions

Compound max (nm) max

H2O 167 1480

CH3OH 184 150

CH3Cl 173 200

CH3I 258 365

(CH3)2O 184 2520

CH3NH2 215 600

n * transitions

- Unsaturated compounds containing atoms

with unshared electron pairs (nonbonding

electrons)

- These result in some of the most intense

absorption in range, 200 – 700 nm

- Unsaturated functional group

- to provide the orbitals

- range: 10 – 100 Lcm-1mol-1

* transitions

- Compounds with unsaturated functional

groups to provide the orbitals.

- These result in some of the most intense

absorption in range, 200 – 700 nm

- range: 1000 – 10,000 Lcm-1mol-1

Examples n * and *

C C

O

HH

H

H

* at 180 nm

n * at 290 nm

(A) Absorption by organic compounds

2 types of electrons are responsible:

i. Shared electrons that participate directly

in bond formation ( and bonding

electrons)

ii. Unshared outer electrons (nonbonding

or n electrons)

MOLECULAR SPECIES THAT ABSORB

UV/VISIBLE RADIATION

• The shared electrons in single bonds, C-C or C-H ( electrons) are so firmly held. Therefore, not easily excited to higher E levels. Absorption ( *) occurs only in the vacuum UV region ( 180 nm).

• Electrons in double & triple bonds (electrons) are more loosely held. Therefore, more easily excited by radiation. Absorptions ( *) for species with unsaturated bonds occur in the UV/VIS region ( 180 nm)

Absorption by organic compounds

CHROMOPHORES

Unsaturated organic functional

groups that absorb in the UV/VIS

region.

Absorption by organic compounds

Typical organic functional groups

that serve as chromophores Chromophores Chemical structure Type of transition

Acetylenic -CC- *

Amide -CONH2 *, n *

Carbonyl >C=O *, n *

Carboxylic acid -COOH *, n *

Ester -COOR *, n *

Nitro -NO2 *, n *

Olefin >C=C< *

• Groups such as –OH, -NH2 & halogens

that attached to the double bonded atoms

cause the normal chromophoric absorption

to occur at longer (red shift).

Absorption by organic compounds

AUXOCHROME

Effect of Multichromophores

on Absorption

• More chromophores in the same molecule

cause bathochromic effect ( shift to longer )

and hyperchromic effect (increase in

intensity).

• In conjugated chromophores, * electrons are

delocalized over larger number of atoms.

This cause a decrease in the energy of *

transitions and an increase in due to an

increase in probability for transition.

• Factors that influenced the :

i) Solvent effects (shift to shorter : blue

shift)

ii) Structural details of the molecules

Absorption by organic compounds

Absorption spectra for typical organic

compounds

• Hypsochromic shift (blue shift)

- Absorption maximum shifted to shorter

• Bathochromic shift (red shift)

- Absorption maximum shifted to longer

Important terminologies

Nature of Shift Descriptive Term

To Longer Wavelength Bathochromic

To Shorter Wavelength Hypsochromic

To Greater Absorbance Hyperchromic

To Lower Absorbance Hypochromic

Terminology for Absorption Shifts

(B) Absorption by inorganic species

• Involving d and f electrons absorption

• 3d & 4d electrons

- 1st and 2nd transition metal series

e.g. Cr, Co, Ni & Cu

- Absorb broad bands of VIS radiation

- Absorption involved transitions between filled and unfilled d-orbitals with energies that depend on the ligands, such as Cl-, H2O, NH3 or CN- which are bonded to the metal ions.

Absorption spectra of some transition-metal ions and rare

earth ions

Most transition metal ions are colored (absorb in UV-VIS) due to d d

electronic transitions

• 4f & 5f electrons

- Ions of lanthanide and actinide elements

- Their spectra consists of narrow, well-

defined characteristic absorption peaks.

Absorption by inorganic species

(C) Charge transfer absorption

Absorption involved transfer of electron from the donor to an orbital that is largely associated with the acceptor.

an electron occupying in a or orbital (electron donor) in the ligand is transferred to an unfilled orbital of the metal (electron acceptor) and vice-versa.

e.g. red colour of the iron (III) thiocyanate complex

Absorption spectra of aqueous charge transfer

complexes

• The fundamental law on which absorption

methods are based on Beer’s Law (Beer-

Lambert Law).

Quantitative Analysis

• You must always attempt to work at the

wavelength of maximum absorbance

(max).

• This is the point of maximum response, so

better sensitivity and lower detection limits.

• You will also have reduced error in your

measurement.

Measuring Absorbance

• Calibration curve method

• Standard addition method

Quantitative Analysis

• Calibration curve method

- A general method for determining the

concentration of a substance in an

unknown sample by comparing the

unknown to a set of standard sample of

known concentration.

Standard Calibration Curve

Ab

so

rban

ce

How to measure the concentration of unknown?

• Practically, you have measure the absorbance of your

unknown. Once you know the absorbance value, you can just

read the corresponding concentration from the graph.

How to produce standard calibration curve

• Prepare a series of

standard solution with

known concentration.

• Measure the absorbance of

the standard solutions.

• Plot the graph Abs vs

concentration of std.

• Find the “best’ straight line.

Stock solution

100 ppm

Calibration standard

Absorb

ance

• The slope of the line, m:

m = y2 – y1

x2 – x1

• The intercept, b:

b = y – mx

• Thus, the equation for the least-square line is:

y = mx + b

Concentration, x y = mx + b

5

10

15

20

25

• From the least-square line equation, you can calculate

the new y values by substituting the x value.

• Then plot the graph.

Standard addition method

- used to overcome matrix effect

- involves adding one or more increments

of a standard solution to sample aliquots

of the same size.

- Each solution is diluted to a fixed volume

before measuring its absorbance.

Absorb

ance

Standard Addition Plot

How to produce standard

addition curve?

1. Add same quantity of unknown sample to a series of flasks.

2. Add varying amounts of standard (made in solvent) to each

flasks, e.g. 0, 5, 10, 15 mL).

3. Fill each flask to line, mix and measure.

Standard Addition

Methods

Single-point standard

addition method Multiple standard

addition method

Standard addition

- if Beer’s Law is obeyed,

A = bVstdCstd + bVxCx

Vt Vt

= kVstdCstd + kVxCx

k is a constant equal to b

Vt

Standard Addition

- Plot a graph: A vs Vstd

A = mVstd + b

where the slope m and intercept b are:

m = kCstd ; b = kVxCx

• Cx can be obtained from the ratio of these

two quantities: m and b

b = kVxCx

m kCstd

Cx = bCstd

mVx

• 10 ml aliquots of raw-water sample were pipetted into 50.0 ml volumetric flasks. Then, 0.00, 5.00, 10.00, 15.00 and 20.00 ml respectively of a standard solution containing 10 ppm of Fe3+ were added to the flasks, followed by an excess of aqueous potassium thiocyanate in order to produce the red iron-thiocyanate complex. All the resultant solutions were diluted to volume and the absorbance of each solution was measured at the same.

Example:

Vol. of std added

(ml)

Absorbance

(A)

0 0.215

5.00 0.424

10.00 0.625

15.00 0.836

20.00 1.040

The results obtained:

Calculate the concentration of Fe3+ (in ppm)

in the raw-water sample

0

0.2

0.4

0.6

0.8

1

1.2

-10 -5 0 5 10 15 20 25

Ab

so

rban

ce

Vol. of std

Absorbance vs Vol. of std added

Slope, m = 0.0382

b = 0.24

(Vstd)0 = -6.31 ml

Note: From the graph, extrapolated value represents the volume of

reagent corresponding to zero instrument response.

• The unknown concentration of the analyte

in the solution is then calculated:

Csample = -(Vstd)0Cstd

Vsample

Cx = bCstd

mVx

The chromium in an aqueous sample was determined by pipetting

10.0 ml of the unknown into each of 50.0 mL volumetric flasks.

Various volumes of a standard containing 12.2 ppm Cr were added

to the flasks, following which the solutions were diluted to the mark.

SELF-EXERCISE

Volume of

unknown (mL)

Volume of

standard (mL)

Absorbance

10.0 0.0 0.201

10.0 10.0 0.292

10.0 20.0 0.378

10.0 30.0 0.467

10.0 40.0 0.554

i) Plot a suitable graph to determine the concentration of Cr in the

aqueous sample.

The portion of the EM spectrum from 400-800 is

observable to humans- we (and some other mammals)

have the adaptation of seeing color at the expense of

greater detail.

400 500 600 800 700

, nm

Violet 400-420

Indigo 420-440

Blue 440-490

Green 490-570

Yellow 570-585

Orange 585-620

Red 620-780

Visible Spectroscopy

When white (continuum of λ)

light passes through, or is

reflected by a surface, those λs

that are absorbed are

removed from the transmitted

or reflected light respectively.

What is “seen” is the

complimentary colors (those

that are not absorbed).

This is the origin of the “color

wheel”.

Visible Spectroscopy

Organic compounds that are “colored” are typically those with extensively conjugated systems (typically more than five). Consider b-carotene.

b-carotene, max = 455 nm

λmax is at 455 nm – in the far blue

region of the spectrum . This is

absorbed.

The remaining light has the

complementary color of orange.

Visible Spectroscopy

λmax for lycopene is at 474 nm – in the near blue region of

the spectrum this is absorbed, the compliment is now red.

λmax for indigo is at 602 nm – in the orange region of the

spectrum. This is absorbed, the compliment is now indigo!

lycopene, max = 474 nm

NH

HN

O

O

indigo

Visible Spectroscopy