LECTURE 3

In this lecture, you will learn

Molecular species that absorb UV/VIS radiation.
Absorption process in UV/VIS region in terms of its electronic transitions.

Important terminologies in UV/VIS spectroscopy.

Organic compounds

Inorganic species

MOLECULAR SPECIES THAT ABSORB UV/VISIBLE RADIATION

Charge transfer

ABSORBING SPECIES
Organic compound
Chemical compound whose molecule contain carbon.

Example C6H6, C3H4

Inorganic species
Chemical compound that does not contain carbon.

Example transition metal, lanthanide and actinide

elements

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

the other is an electron acceptor. Example Iron(III) thiocyanate complex

NOTE: Transition metals - groups IIIB through IB

UV-VIS ABSORPTION
In UV/VIS spectroscopy, molecules which absorbed

the EMR in this region will result in transitions between electronic energy levels.

Molecular Absorption
In molecules, instead consist of electronic level, it also comprise of vibrational and rotational sub-levels. Transitions between these molecular energy levels will result in band spectra.

TYPE OF TRANSITIONS
There are 3 types of electronic transitions

, and n electrons 2. d and f electrons 3. Charge transfer electrons

1.

What is ,
H + O + H

and n electrons?
single covalent bonds ()
H O H or H O H

lone pairs(n)
O C O or O C O

double bonds ()

or

triple bond ()

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 wavelength. Therefore, alkanes and other

saturated compounds (compounds with only single bonds) do not absorb UV radiation.
As a result, Alkanes such as hexane, C6H14 are frequently very useful as transparent solvents for the study of other molecules.

Pi () electron
Electrons involved in double and triple

bonds (unsaturated). These bonds involve a bond. bond exists in alkenes, alkynes, conjugated
olefins and aromatic compounds. Electrons in bonds are excited easily. These compounds commonly absorb UV or visible photons.

Organic molecules containing bonds

H3C
CH2CH3

C
H

Propyne (alkynes)

H C C H

C C C C H

H H C C H C H C

H H

Ethylbenzene

Benzene

(aromatics & alkenes)

1,3-butadiene (conjugated olefins)

Lone pair (n) electron

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 are nonbonding. Compounds that contain n electrons absorb UV/VIS radiation.

Organic molecules with non-bonding electrons

NH2

H3C
C R
aminobenzene Carbonyl compound

H C C H

Br

2-bromopropene

Electronic energy levels diagram

Unoccupied levels

Occupied levels

Graphical representation of electrons in orbitals

* *
Atomic orbital Atomic orbital
Unoccupied levels

Energy

Occupied levels

Molecular orbitals

16

From the molecular orbital diagram, there are several possible electronic transitions that can occur. Each of them at different relative energy.
* *
n * alkanes * carbonyls * Unsaturated compouds. * O, N, S, halogens

Energy

* carbonyls

17

Absorption by Organic Compounds

Absorption of UV/Vis by organic compounds

requires that the energy absorbed corresponds to a jump from occupied orbital unoccupied orbital. Generally, the most probable transition is from the

highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO).

* transitions

Never observed in normal UV/Vis work.

The maximum absorption are below than 150 nm. The energy required to induce a * transition is too big (see the arrow in energy level diagram). This type of absorption corresponds to breaking

bonds such as C-C, C-H, C-O, C-X, .

vacuum UV region

* transitions

Saturated compounds must contain atoms with unshared electron pairs.

Compounds containing O, S, N and halogens can

absorb via this type of transition. Absorptions are typically in the 150 -250 nm region and are not very intense. range: 100 3000 Lcm-1mol-1

Some examples of absorption due to n * transitions

Compound H2O CH3OH CH3Cl CH3I (CH3)2O CH3NH2 max (nm) 167 184 173 258 184 215 max 1480 150 200 365 2520 600

* transitions

Unsaturated compounds containing atoms with unshared electron pairs.

These result in some of the most intense

absorption in 200 700 nm region. range: 10 100 Lcm-1mol-1

* transitions

Unsaturated compounds to provide the orbitals.

These result in some of the most intense absorption in 200 700 nm region. range: 10oo 10,000 Lcm-1mol-1

Some examples of absorption due to

and

* transitions

CHROMOPHORES
Unsaturated organic functional groups that absorb in the UV/VIS region.

AUXOCHROME
Groups of atoms such as OH, -NH2 & halogens that attached to the double bonded atoms cause the normal chromophoric absorption to occur at longer (red shift).

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.

Other Factor that Influenced Absorption

Factors that influenced the : 1. Solvent effects (shift to shorter : blue shift) 2. Structural details of the molecules.

Important terminologies

hypsochromic shift (blue shift)

- Absorption maximum shifted to shorter

bathochromic shift (red shift)

- Absorption maximum shifted to longer

Terminology for Absorption Shifts

Nature of Shift To Longer Wavelength To Shorter Wavelength To Greater Absorbance To Lower Absorbance Descriptive Term Bathochromic Hypsochromic Hyperchromic Hypochromic

Absorption by Inorganic Species

Involving d and f electrons absorption.

1. 3d & 4d electrons
1st and 2nd transition metal series for example 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

Absorption by Inorganic Species

2. 4f & 5f electrons
Ions of lanthanide and actinide elements. Their spectra consists of narrow, well-defined characteristic absorption peaks.

Typical absorption spectra for lanthanide ions

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

INSTRUMENTATION

Important components in UV-Vis Spectrophotometer

1 2

Source lamp
UV region:
- Deuterium lamp; H2 discharge tube

Sample holder

selector

Detector

Signal processor & readout

Quartz/fused silica

Prism/monochromator

Phototube, PM tube, diode array

Visible region:
- Tungsten lamp Glass/quartz

Prism/monochromator

Phototube, PM tube, diode array

UV-VIS INSTRUMENT
1. Single beam 2. Double beam

Single beam instrument

Single beam instrument

Only 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 results 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 between 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

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 Compensate for wide variations in source intensity with . Continuous recording of transmittance or absorbance spectra.

Quantitative Analysis
The fundamental law on which absorption methods are based on Beers law (Beer-Lambert law).

How to measure the absorbance?

1. You must always attempt to work at the

wavelength of maximum absorbance, max

2. This is the point where it has the maximum response due to better sensitivity and lower detection limits. 3. You will also have reduced error in your measurement when analysed the samples and standards at max .

Quantitative Analysis
1. Calibration curve method 2. Standard addition method

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 solution of known concentration.

Standard Calibration Curve

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?

A

1. Prepare a series of standard

solution with known concentration. 2. Measure the absorbance of the standard solutions. 3. Plot the graph Absorbance vs Concentration of the standard solutions. 4. Find the best straight line by using least-squares method.

Finding the Least-Squares Line

Concentration xi 5 10 Absorbance yi 0.201 0.420 xi2 yi2 xiyi

15
20 25

0.654
0.862 1.084

xi
N=5

yi

xi2

yi2

xiyi

N is the number of points used

The calculation of the slope and intercept is simplified by defining three quantities Sxx, Syy and Sxy. Sxx Syy Sxy = (xi x)2 = xi2 ( xi)2
N

(1) (2)

= (yi y)2 = yi2 ( yi)2

N

= (xi x) (yi y)2 = xiyi xi yi (3)

N

 The slope of the line, m:

m = Sxy Sxx The intercept, b: c = y mx Thus, the equation for the least-squares line is y = mx + c

Concentration, x 5 10 15 20 25

y = mx + c

From the least-squares line equation, you can calculate the new y values by substituting the x value. Then plot the graph.

Most linear regression implementations have an option to force the line through the origin, which means forcing the intercept of the line through the point (0,0). This might seem reasonable, since a sample with no detectable concentration should produce no response in a detector, but must be used with care.

HOWEVER, forcing the plot through (0,0) is not always recommended since most curves are run well above the instrumental limit of detection (LOD). Randomly, adding a point (0,0) can skew the curve because the instruments response near the LOD is not predictable and is rarely linear. As illustrated next page, forcing a curve through the origin can lead to bias results.

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.

Standard Addition Curve

Absorbance

Concentration, ppm

How to produce standard addition curve?

1. Add same quantity of unknown sample (spike) to a series

of volumetric flasks.
2. Add gradual amounts of standard solution to each

volumetric flask. For example 0 mL, 5 mL , 10mL , 15mL.

3. Dilute each flask to calibration mark, mix and measure the

absorbance.

Standard Addition Methods

Single-point standard addition method (Focus on this) Multiple additions method

Standard Addition Methods

If Beers law is obeyed,

A = bVstdCstd + bVxCx Vt Vt = kVstdCstd + kVxCx A = (kCstd)Vstd + kCxVx

k is a constant equal to b/Vt

Standard Addition Methods

Plot a graph of A vs Vstd

A = (kCstd)Vstd + kCxVx Y= mX + C
where the slope m and intercept C are

m = kCstd C = kCxVx

Cx can be obtained from the ratio of these two quantities m and C

C m
Cx

= kVxCx
kCstd

= CCstd mVx

Standard Addition Methods

For single-point standard addition
Absorbance of diluted sample

A1 = bVxCx Vt A2 = bVxCx + bVsCs Vt Vt

Eq. 1

Absorbance of diluted sample + std

Eq. 2

Standard Addition Methods

For single-point standard addition Dividing the 2nd equation by the first & then rearrange it will give.

Cx =

A1 Cs Vs (A2 A1 ) Vx

Standard Addition (single point addition)

A 2.00-mL urine specimen was treated with reagent to generate a color with phosphate which then was diluted to 100 mL. Second 2.00mL sample was added exactly 5.00mL of a phosphate solution containing 0.03 mg/mL phosphate then was treated in the same way as the original sample. The absorbance of the first solution was 0.428, while the second one was 0.538. Calculate the concentration of phosphate in milligrams per millimeter of the specimen.

Solution

Cx = (0.428) (0.03 mg PO43-/mL) (5.00mL) (0.538 0.428)(2.00mL) = 0.292 mg PO43- / mL sample

Try These!
The concentration of an unknown chromium solution was determined by pipetting 10.0mL of the unknown into each of five 50.0 mL volumetric flasks. Various volumes of a standard containing 12.2 ppm chromium were added to the flasks and then the solutions were diluted to the mark.
Standard, mL 0.0 10.0 Absorbance 0.201 0.292

20.0
30.0

0.378
0.467

40.0

0.554

Determine the concentration of chromium (in ppm) in the unknown.

Visible Spectroscopy
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

700

800

, nm Violet Indigo Blue Green Yellow Orange Red 400-420 420-440 440-490 490-570 570-585 585-620 620-780

Visible Spectroscopy
When white (continuum of ) light passes through, or is reflected by a surface, those s 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

max 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
lycopene, max = 474 nm
O H N N H O

indigo

max for lycopene is at 474 nm in the near blue region of the spectrum this is absorbed, the compliment is now red. max for indigo is at 602 nm in the orange region of the spectrum. This is absorbed, the compliment is now indigo!

Visible Spectroscopy
One of the most common class of colored organic molecules are the azo dyes:
N N

EWGs

EDGs

From our discussion of di-subsituted aromatic chromophores, the effect of opposite groups is greater than the sum of the individual effects more so on this heavily conjugated system. Coincidentally, it is necessary for these to be opposite for the original synthetic preparation!

These materials are some of the more familiar colors of our environment
NO2

HO O3S N N

N N OH

H2N

N N NH2

SO3
Fast Brown

Sunset Yellow (Food Yellow 3)

Para Red

The colors of M&Ms

Bright Blue
Common Food Uses Beverages, dairy products, powders, jellies, confections, condiments, icing.

Royal Blue
Common Food Uses Baked goods, cereals, snack foods, ice-cream, confections, cherries.

Orange-red
Common Food Uses Gelatins, puddings, dairy products, confections, beverages, condiments.

Lemon-yellow
Common Food Uses Custards, beverages, ice-cream, confections, preserves, cereals.

Orange
Common Food Uses Cereals, baked goods, snack foods, ice-cream, beverages, dessert powders, confections