Anda di halaman 1dari 28

An Introduction to Ultraviolet/Visible Molecular Absorption Spectrometry

Click to edit Master subtitle style 3/19/12

The need for instrumental analysis

How to choose an analytical method? How good is measurement? How reproducible? - Precision How close to true value? Accuracy/Bias How small a difference can be measured? - Sensitivity What range of amounts? - Dynamic Range 3/19/12

SPECTROSCOPY (spectrochemical analysis)


An analytical method based on the interaction of the electromagnetic radiation and the matter (analyte). The following points should be revised: Electromagnetic radiation : ( nature , characteristics , energy , interaction with matter . etc. ) Molecular absorption Please, refere to the printed papers . 3/19/12

Energy and Electromagnetic Radiation


Light is a particle = Photon E (energy of photon) = hv h (Plancks constant) = 6.626 x 10-34 J s c = v , v = c/ E = hc/ = hcv* V* = wavenumber
3/19/12

Electromagnetic Radiation and Its Absorption by Molecules

3/19/12

UV and Vis light cause only two kinds of electronic transition

3/19/12

Absorbing Species
Absorbing species containing s, p, and n electrons (organic compounds) . Ground state + e.m.r. energy = excited state

3/19/12

Absorbing Species
A chromophore is the part of a molecule which absorbs UV or visible light

3/19/12

Chromophores

3/19/12

Cont.
Such a table illustrates examples of chromophores with the type of electronic transition taking place in molecules when excited by absorption of electromagnetic radiation . Its only for use to expect the properties of organic compound including one or more of these chromophores .
3/19/12

Colorimetry

Analytical methodColor based on Wavelengt Color observed Absorbed absorption of visible light(400- h (nm) GreenViolet 380-420 800nm) yellow yellow Violet-blue 420-440 1. Colored Analytes orange Blue 440-470 2.Indicators/Dyes/Chromogenic 470-500 red Blue-green Substrates purple green 500-520 (e.g. Serum Iron Determination) violet Yellow520-550 green Colors of VisibleViolet-blue yellow Light 550-580
blue 3/19/12 orange 580-620 620-680 Blue-green Red

Requirements for substances to be measured colorimetricaly:


Substance must be coloured e.g CuSO4, organic dyes,. 2-If the substance to be analysed is colourless, it must react with certain reagent (known as chromogen) to produce equivalent coloured product. 3-If there is no suitable chromogen, the substance must be converted to a certain 3/19/12 derivative which has a suitable chromogen.
1.

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

3/19/12

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 3/19/12 rapid and quantitative

Requirements for substances to be measured colorimetricaly:(cont.)

4-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 . Esters are first converted to hydroxamic acid derivative through the reaction with hydroxylamine. Hydroxamic acid derivative gives purple color on addition of ferric 3/19/12

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. 3/19/12 5-It must produce only one color of

U.V.-Vis. Absorbance measurement


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). 3/19/12 It is characteristic for each molecule

U.V.-Vis. Absorbance measurement

3/19/12

Factors affecting absorption spectrum


A) Presence of chromophores and Auxochromes .
1)Chromophres : Are unsaturated groups responsible for - * and n- * electronic transitions. e.g. C=C , C=O , N=N and N=O ( 200nm-800nm) . B)Bathochromic & Hypsochromic shift . -Bathochromic shift (or red hift) It is the shift of max to a longer wavelength due to substitution with certain functional groups 3/19/12 (e.g. OH and NH2), when two or more

Cont.

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
3/19/12

Cont.

2) 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
3/19/12

Cont.
Change in absorption spectrum :

3/19/12

Factors affecting absorption spectrum (cont.)

C) Conjugation Increase in conjugation,


3/19/12

Cont.

phenol

aniline

3/19/12

Cont.
Anilines spectrum exhibits bathochromic shift and hyperchromic effect in alkaline medium due to its conversion to the quinonoid species . Its spectrum in acid medium exhibit hypsochromic shift and hypochromic effect due to its conversion to the benzenoid species.

3/19/12

Isosbestic point

3/19/12

Cont.
-At different pH, the spectrum will be shifted to different max but 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 3/19/12 the isosbestic point. out at

Exercise

Sketch the spectrum of aniline in a acidic medium (0.1 M HCl) , neutral medium , alkaline medium ( NaOH) .

3/19/12