Lectures UV Spectroscopy

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Lecture III 2) Chromophores and Auxochromes . 3) Bathochromic shift and Hypsochromic shift . 1) Absorption Spectrum .

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Page 1: Lectures UV Spectroscopy

Lecture III

2) Chromophores and Auxochromes .

3) Bathochromic shift and Hypsochromic shift .

1) Absorption Spectrum .

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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.

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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)

Are saturated groups posses unshared electrons, and does not absorb in near UV or visible radiations e.g. OH,NH2.


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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

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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

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Lecture IV

1)Effect of pH on absorption spectrum.

2)Polychromatic and Monochromatic light.

3)Theory of light absorption

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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.






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


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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.

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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

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Lecture VI

2-Colorimetry .

1-Isosbestic point

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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.

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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.

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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

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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.

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3 + Fe2+






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.

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-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 .


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.

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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

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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.

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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

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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