Spectrophotometry

Spectroscopy is the study of

interaction of spectrum of light with

a substance to be analysed, for its

identification (i.e qualitative analysis)

as well as determination of its

amount (i.e quantitative analysis).

Light It is one of the different forms of radiant

energy, such as, sunlight, radio waves and X-rays .

Thy are called electromagnetic radiations (EMR) due presence of both electric and magnetic components

Dual nature of light

Light exhibits wave property during its propagation and energy particle during its interaction with matter. The double nature of light (waves and particles) is known as dualism.

EMR display the property of continuous waves and can be described by the characteristics of wave motion.

Such wave motion in conveniently classified according to the wavelength.

1. Wave property

It is the linear distance measured along the line of propagation between crest of one wave to the next.

Unit of Length

Micron () = 1 x10-6m = 1 x 10-4cm = 1x 10-3 mm.

Millimicron (m) = or nanometer (nm) = 1 x 10-9m = 1 x 10-7 cm = 1 x 10-6mm.

Angstrom (Ao) = 1 x 10-10m = 1 x 10-8cm = 1 x 10-7 mm.

Wavelength ()

Frequency []

is the number of waves/ second or number of cycles occurring/ second (CPS) or Hertz (Hz) or waves vibration/ second.

Relation between &

= C /

where c is the velocity of light cm/s.

C= 3.0 x 1010 cm/s C =

Wave number ( `) is the number of waves/ cm

`= 1/ Its unit is cm-1

2-Particle property (light as energy)

Light consist of energy packets, known as photons.

The energy (E) of photons is proportional to the frequency i.e related to c and . It can be expressed by max plank relation:

E = h

where h = max plank constant = 3.63 x 10-27 erg., sec.)

i.e E

or E

( = C / )

1/

Therefore energy of a beam of EMR increases as wave length decreases.

The shorter the wave length, the greater the energy of the photons and the more powerful the radiation.

U.V range (200nm-400nm) which contain shorter , carrying more energy photon than a beam of visible range .

Visible range (400nm-800nm) having high energy more than I.R range (>800nm).

ExampleExample:: The The λλ of the sodium D line is 589nm . What are of the sodium D line is 589nm . What are

the frequency(the frequency( ) and wave umber ( ) and wave umber ( `) `)

SolutionSolution:: = C/= C/λλ

C=3.0 x 10C=3.0 x 108 8 m/s λnm----λm (nm x 10-9 ) = = 3x103x1088m/sm/s / / 589nm x 10589nm x 10-9-9 == 5.09x10 5.09x101414 s s-1-1

`= 1 / `= 1 / λλ ((`= cm`= cm-1-1 ) ) λλ nm----- nm----- λλ cm ( nm x 10 cm ( nm x 10-7-7 cm ) cm )

`= `= 11 // 589 x10589 x10-7-7 == 1.7 x 10 1.7 x 1044 cm cm-1-1

Second Second LecturesLectures

For analytical purposes we use the region of I.R, visible and U.V radiations .

-UV radiation region is classified into : far UV from (10nm-200nm) and near UV from ( 200nm-380nm-

-Visible radiation region (380-780nm) consist of colored radiations, which are, violet, indigo, blue, green, yellow, orange and red .

-IR radiation region ( 0.75um-1000um ) is classified into :

near IR , mid IR and far IR .

Interaction of a substance with EMR

E = Es - E g = h

When a molecule interact with radiant energy

The molecule is said to be excited , because the outer valence electrons undergo transition from original energy level ground state (E g) to an excited state (Es).

Excited state Es

Ground state E g

The transition energy is given by the following equation :

1-When the molecule absorb Visible and U.V region .

2-When the molecule absorb I.R region.

Raising the Vibration of molecule (Vibrational transition energy)

3-When the molecule absorb F.I.R and Microwave regions.

Increasing rotation of the molecule (Rotational transition energy)

Raising electrons to a higher energy level (Electronic transition energy )

When a molecule in the ground state absorbs EMR, 3 energy state transition will take place. These types of transition are :

1) Electronic 2) Vibrational 3) Rotational .

The total energy E total = E electronic + E vibrational + E rotational

The relative value of E electronic : E vibrational: E rotational is :

10000 : 100 : 10

(1) Photons of Far IR and Microwave spectrum (E↓) cause only rotation of the molecules.

(2) Photons of IR spectrum (E >E far IR and microwave ) cause vibration and rotation of the molecule

(3) Photons of UV-Visible spectrum (E↑>EIR >E far

IR) cause electronic transition which is accompanied by vibrational and rotational transion .

The relation between structure and energy The relation between structure and energy absorbedabsorbed

The energy absorbed by a molecule in The energy absorbed by a molecule in the ground statethe ground state is dependent on the nature of the bonds within a is dependent on the nature of the bonds within a moleculemolecule

-The outermost electrons in organic molecules may be:

strong (σ) bonds, weaker () bonds or nonbonding (n)1-Sigma () electrons: they are bonding electrons posses the lowest energy level ( the most stable single bonds).

2-Pi () electrons :the bonding electrons constituting the pi bonds (the weaker double or treble bonds)

3-Non-bonding (n) : electrons: don’t participate in bonding, they usually occupy the highest level of ground state (hetero atoms like N,O,S )

In the excited state ;

--electrons occupy the anti bonding * level, and the transition is termed - * .

-while n electrons occupy either * or *,and the transition is termed n- * or n- *

- electrons occupy an anti bonding energy level denoted as * and the transition is termed -* transition.

-Compounds containing only -electrons ( →σ*

transition) are the saturated hydrocarbons which absorb at <180nm in the far UV.

-n-electrons absorption in saturated compounds (n→σ* transition ) containing hetero atoms or halogens.

-Alcohols and ethers absorb at wavelength shorter than

185nm and so they are useful as common solvents at > 200nm.

-They are transparent in the near UV (200-300nm) making them ideal solvents for other compounds to be studied in this region.

-The majority of these compounds show no bsorption in the near UV,eg. Methanol at 177nm, tri ethylamine at 199nm or chloroform at 173nm.

-- * transition in unsaturated compounds containing double or triple bonds are easily exited than in single covalent bonds.

-Also , the energy required for - * in conjugated compounds is smaller than in non-conjugated ones.

-So conjugated compounds absorb at longer wave length than un-conjugated one .

-n→ * transition occur in compounds containing

non-bonding electrons adjacent to unsaturated centers

(double or triple bonds)

Suggest which of these compounds Suggest which of these compounds absorb in UV-Visible spectrumabsorb in UV-Visible spectrum ??

CompoundCompound: T: Type of transitionype of transition: A: Answernswer

1) CH1) CH33-CH-CH33 : :

2) CH2) CH22=CH=CH2 2 ::

3) CH3) CH33-O-H : -O-H :

4) CH4) CH33-C=O :-C=O : | | CHCH33

Lecture III

2) Chromophores and Auxochromes .

3) Bathochromic shift and Hypsochromic shift .

1) Absorption Spectrum .

1) Absorption spectrum

according to the electronic transition that occur in each organic molecule, absorption spectrum is obtained by plotting Absorbance (A) as a function of wavelength ().

It has characteristic shape with the of maximum absorbance (max).

It is characteristic for each molecule according to its structure and the type of transitional energy

Therefore it is used for identification of a chemical substance (qualitative analysis). Also max is used for quantitative measurement, in order to increase sensitivity and to minimize error of the analytical method.

2) Chromophores and Auxochromes2) Chromophores and Auxochromes Chromophres :

Are unsaturated groups responsible for - * and n→ * electronic

transitions. e.g. C=C , C=O , N=N and N=O ( 200nm-800nm)

3)Bathochromic & Hypsochromic shift

It is the shift of max to a longer wavelength due to substitution with certain functional groups (e.g. –OH and –NH2), when two or more chromophores are present in conjugation, change in pH and effect of the medium (solvent).

- Hypsochromic shift (or blue shift)It is the shift of max to a shorter wavelength due to removal of conjugation by changing pH or polarity of the solvent.

-Bathochromic shift (or red hift)

-Auxochromes

Are saturated groups posses unshared electrons, and does not absorb in near UV or visible radiations e.g. OH,NH2.But when attached to chomophoric molecule, increase both its

wave length and intensity of absorption maximum .

Because auxochrome inters into resonance interaction with the chromophore , thus increase the extent of conjugation, shift the absorption maximum to longer wave length

- Hyperchromic effect

an increase in the intensity of absorption usually due to introduction of an auxochrome - Hypochromic effect

It involves a decrease in the intensity of absorption

Example 1): effect of conjugation on absorption spectrum

Increase in conjugation, increas absorbance of light to higher , bathochromic shift with hyperchromic effect.

Changes in

Absorption spectrum

Lecture IV

1)Effect of pH on absorption spectrum.

2)Polychromatic and Monochromatic light.

3)Theory of light absorption

Example 2) Effect of pH

The spectra of compounds containing acidic (phenolic-OH) or basic (-NH2) groups are dependent on the pH of the medium.

OH O

O

..:

..:

OH-

H++ H

+

-

-

The U.V spectrum of phenol in acid medium, benzenoid form while in alkaline medium is the phenate anion ,quinonoid formThe free pair of of

electrons of O2 increasing the elocalization of the -electrons, leading to the formation of conjugated system. So , electrons become more

energetic and need less energy to be excited, therefore absorb longer bathochromic shift ; red shift) with hyperchromic effect

Phenol

Aniline

NH3NH2

NH2

:

OH-

H+

+

+

-

Its spectrum exhibits bathochromic shift and hyperchromic effect in alkaline medium due to its conversion to the quinonoid speciesWhile in acid medium (anilinium ) lost the free pair electrons of N decrease the conjugation .

Its spectrum in acid medium exhibit hypsochromic shift and hypochromic effect due to its conversion to the benzenoid species.

Polychromatic light

A beam of light containing several wavelengths , e.g. white light

Monochromatic light

A beam of light containing radiation of only one discrete wavelength

29

Theory of light absorption :

Some is absorbed (I a), reflected (I r), transmitted (It), refracted (If) and scattered (Is).Is = zero for clear solution, while If and Ir may be

canceled by means of control cuvette containing the solvent in which the substance to be anaylsed is dissolved Therefore, under experimental conditions.

Io = Ia + It or Ia = Io -

It

When a monochromatic light having intensity (Io) is allowed to pass through absorbing medium ;

1-Beer-Lambert Law

Lecture V

2-Absorptivity ,

Molar absorptivity and

A1%.1cm

The diagram below shows a beam of monochromatic radiation of radiant power Io, directed at a sample solution. Absorption takes place and the beam of radiation leaving the sample has radiant power I.

Absorbance A = log10 I0 / It

A = log10 1 / T

A = log10 100 / %T A = 2 - log10 %T 

Transmittance T = It / Io

% Transmittance %T = 100 x T

The relationship between absorbance and transmittance is illustrated in the following diagram:

So, 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.

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

log Io/It b (thickness )

log Io / It = K b

lamberts’ law:

When a monochromatic light enter absorbing medium, its intensity is decreased exponentially with the

increase of thickness of the absorbing medium (i.e solution) (b) at constant concentration (C)

C

b

It

C

b

Log Io/It

A

log Io/It C (concentration) log Io / It = K C

Beers’ law

When a monochromatic light enter absorbing medium, its intensity is decreased exponentially with the increase of concentration of the absorbing medium (C) at constant pathlength (b)

bC

It

bC

Log Io/It

A

which is the absorbance, when thickness of solution is unity (1cm) and concentration is unity (1gm/L)

Absorbance (A ) = log Io/It

log Io/It b C

log Io/It = a b C

A = a b C

a: is a constant, known as absorptivity

log Io/It b C

Beer-lambert’s law:

(a) is known as molar absorptivity or epsilon ()

If the unit of concentration is 1M

Molar absorptivity or epsilon ()

i.e. conc.= mol/L

Its unit is L mol-1 cm-1

As, a=A/b c =A/1cm . gm/L = L.gm-1.cm-1

molar absorptivity , =A/b c= A/1cm.mol/L

(mol=gm/ mol. weight)

i.e. Its unit = L.MWt.cm-1gm-1.

= a x mol. wt a = / mol.wt

If unit of concentration is 1% ( i.e. 1gm/100 ml )

A (1% - 1cm):

A (1% - 1cm) = A/ 1cm.gm% = 100. cm-1.gm-1.

a= L.gm-1. cm-1

A (1% - 1cm) = a x 10 a= A (1% - 1cm) / 10

(a = / mol. Wt = A (1% - 1cm) / 10 )

= A (1% - 1cm) x mol. Wt / 10

A (1% - 1cm) = x 10 / mol. Wt

Problem 1: A sample solution give absorbance equals 0.6 ,

A1% 1cm = 1030 and its molecular weight = 147.

Calculate the sample concentration in Mol/L

Solution 1:

A =ε b c = 15141 x 1 x C 0.6 =15141 x C

C = 0.5 / 15141 = 3.96 x 10-5 mol / L

ε = A 1% 1cm x M.wt / 10 = 1030 x 147 / 10 = 15141 litre.cm-1.mol-1

Both and A 1%, 1cm are characteristic for each substance at the same max , pH and type of solvent and are used for quantitative purpose

Lecture VI

2-Colorimetry .

1-Isosbestic point

Isosbestic point

-At different pH, the spectrum will be shifted to different maxbut all spectra intersect at certain which is known as isosbestic point

- At isosbestic point, the same absorbance is given for the same concentration at different pH,i.e. absorbance is not pH dependent but concentration dependent-Thus solution ; its max affected by pH , must be buffered at specific pH or measurements are carried out at the isosbestic point.

Colorimetry

When white light passes through a colored substance, a characteristic portion of the mixed wavelengths is absorbed.

Complementary colors are diametrically opposite each other. Thus, absorption of 420-430 nm light renders a substance yellow, and absorption of 500-520 nm light makes it red.

The remaining light will then assume the complementary color to the wavelength(s) absorbed.

Colored substances appear colored because they selectively absorbed some of wavelengths of visible light and transmitted other wavelengths or colors (apparent color),

Red substances absorb the blue- green wavelengths from the visible region, so the transmitted light appears red

Blue substances absorb the yellow wavelengths, so the transmitted light appears blue.

wavelength region, nm color complementary color

400-435 Violet Yellow-green

435-480 Blue Yellow

480-490 Blue-green Orange

490-500 Green-blue Red

500-560 Green Purple

560-580 Yellow-green Violet

580-595 Yellow Blue

595-650 Orange Blue-green

650-750 Red Green-blue

2-If the substance to be analysed is colourless, it must react with certain reagent (known as chromogen) to produce equivalent coloured product.

1.Substance must be coloured e.g CuSO4, organic dyes,….

Requirements for substances to be measured colorimetricaly:

3-If there is no suitable chromogen, the substance must be converted to a certain derivative which has a suitable chromogen.

QuizI)Discuss shortly

-Interaction of a substance with EMR

-Factors affecting absorption spectrum

II) Solve the following problem

1-A 5.00x10-4M sample solution is measured in a cell with 1 cm bath length ; its absorbance at 592nm equals 0.446 .a-What is the molar absorptivity at 582nm.

If a solution of unknown concentration of the same sample has an absorbance 0.125 at the same wave length. b-What is its concentration

2-Calculate the wave legnth in um , and in Angestron

3-Calculate the frequancy and energy of this wave length

III) Complete the answer in Exercise 1

N

N

3 + Fe2+

N

N

Fe

2+

3

1.Orthophenanthrolene reacts with ferrous (Fe2+)in buffered medium (acidic pH) to produce intense red color.

2-If the substance to be analysed is colourless, it must react with certain reagent (known as chromogen) to produce equivalent coloured product.

-Esters are first converted to hydroxamic acid derivative through the reaction with hydroxylamine. Hydroxamic acid derivative gives purple color on addition of ferric (Fe3+) due to the formation of iron chelate .

O O

R –C– O Et + H2N – OH

R –C– NH – OH + Et OH

Hydroxamic acid derivative + Fe3+

3-If the sample is colorless and there is no suitable chromogen, the substance must be converted to a certain derivative which can be react with suitable reagent producing color .

then measuring the absorbance at 520nm.

Requirements for ideal chromogen

1-Should be colorless or easily separated from the colored product

2-It Should be selective.3-Its reaction to produce colored product, should be of known mechanism and proceed stoichiometrically.

4-The full development of color must be rapid.

5-It must produce only one color of specified max.

Chromogen is

a compound containing chromophoric group

Requirements for coloured product

1-Should be of intense color, to increase the sensitivity

2-Should be unaffected by pH or the pH must be specified and maintained by suitable buffer or the measurement is carried out at of isosbestic

3-Should be stable with time

4.The reaction of its formation, must be rapid and quantitative.

5-The colored product, should obey Beer-lambert’s law, i.e on plotting A versus C at fixed b, we obtain

straight line passing through the origin.

Instrumentation

The instrument used, usually consists of 5 basic components

1-Radiant energy source.

2-Dispersing system (or monochromator).

3-Sample compartment (cuvette).

4-Detector

5-Recorder (meter).

1. Radiant energy source (source of light)

In visible range

Tungsten lamp

In U.V range Deuterium lamp (D2) (or hydrogen

lamp.) 2. Dispersing system (monochromator)

It convert polychromatic light to monochromatic light (definite range of .)

a) Filters b) Prisms C) Grating

selective absorption of unwanted and transmit the complementary color, which is needed to be absorbed by the sample to be analysed.

Filters may be : gelatin, liquid and tinted glass

Disadvantages:Filter transmit a wide band of 35-50 nm. which is not exactly monochromatic and used only in visible range .

act by

b) Prisms

Act by refraction of light

In visible range

glass prism

In U.V range

quartz or fused silica prism.

As they are transparent to UV lightCannot be used for UV as it absorb all UV light

The dispersion power of the prism is ∝ 1/

i.e. angle of refraction ↑ as ↓

C) Grating

Grating consists of a large number of parallel lines ruled very close to each other on a highly polished surface e.g

aluminum or aluminized glass (600 line/mm).

Each ruled groove functions as a scattering center for light rays falling on its edge.

The grating disperses the light beam into almost single .

. The resolution power of grating is > prism > filter.

It works in both UV and visible spectral region by dispersion.

The scattering power of a grating is ↑ as the number of groves ↑

d) Associated optics are used to control light intensity

Collimating lenses slit of variable width mirrors and diafragms

act as condensers helps to narrow band width alignment of the beam.

They should be suitable for the spectral range, i.e glass for visible range and quartz

or fused silica for U.V range.

3.Sample compartment (Cuvette)

Transparent surface

Opaque surface

It is made of →glass for visible range

quartz or fused silica for U.V range.

4. Detector

a) photocell (Photovoltaic cell) e.g Barrier layer cell

b) Phototube (photomultiplier or photoemissive tube)

Light falling on cell

Transparent metal layer of Ago (Collecting electrode)

Photosenitive semiconductor of selenium

Metal base Plate of iron

-

+

a) photocell (Photovoltaic cell) e.g Barrier layer cell

b) Phototube (photomultiplier or photoemissive tube)

5. Recorder (meter)

Electric signal produced in detector is fed to a sensitive galvanometer,

its scale is graduated in absorbance or/and transmittance units.

Commercial instruments

1. Filter photo-electric colorimeter

2. Compensating two-photocell colorimeter

In this type, fluctuation in intensity of EMR source is automatically cancelled.

3. Prism spectrophotometer