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# Quantitative and Qualitative Spectrophotometry

by Dr. Ibrahim A. Naguib

What is spectroscopy?

Spectroscopy is the study of interaction of spectrum of light
with the analyte, for its qualitative identification and for quantitative determination of its concentration. What is spectrophotometry? Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a

function of wavelength. It is more specific as it deals with
visible light, near-ultraviolet, and near-infrared.

What is the difference between Light and electromagnetic radiations EMR?
Light is one segment of the continuous spectrum known as EMR. EMR includes sunlight, radio waves, cosmic waves and X-rays…etc, which have similar properties and are called electromagnetic radiations (EMR) due to presence of both electric and magnetic components.

Such wave motion in conveniently classified according to the wavelength. . light exhibits wave property and energy particle during its interaction with matter. Wave property EMR display the property of continuous waves and can be described by the characteristics of wave motion.  So what is the Wavelength (λ)? It is the linear distance between crest of one wave to the next. What is the Dual nature of light (Dualism)? When it propagates. The double nature of light (being waves and particles in nature) is known as dualism:  (1).

 Velocity of light {C} Light propagates at the highest known velocity= 300. All kinds of waves propagate at the same speed (they differ in wavelength and frequency). C= λ . 000 Km/sec.   .

  EMR may be visible or invisible Visible radiations form only a small part of the complete EMR. Relation between λ & υ C= λ . Each Definite color is described as monochromatic light. (from 400-800 nm).V. They are subdivided into different colors according to their wavelength (i.e.  or  = C/ λ  b) Wave number (δ): is the number of waves/ cm δ = 1/ λ EMR have the property of frequency which can be expressed by: ..R).    a)Frequency [ (nu)]: is the number of waves/ second. it is polychromatic). cycles / second (CPS). Invisible radiations: which are Ultraviolet (U.. or Hertz (Hz). Etc.) and Infrared (I.

e E α υ or E α 1/ λ α δ  Accordingly.63 x 10-27 erg.e related to C and λ. Particle property Max Planck presented light as matter (with particulate nature) that forms of packets of energy known as (photons). U.) i. e. It is proportional to the frequency i. sec. hence having more energy than visible range. The energy (E) of photons is variable. . and visible range more than I.V range contains shorter λ.R range.g.  It can be expressed by max plank relation: E = h υ (h = Max Plank constant = 3. (2). energy of EMR increases as wavelength decreases.

visible and U.     A beam containing several wavelengths is called polychromatic light. i. the greater the energy of the photons and the more powerful the radiation.e the shorter the wave length. which are.V radiations are used for analytical purposes usually. red.R. we see white light (remember the color wheel!). Day light (visible radiation) consists of colored radiations. The regions of I. yellow. while a beam single wavelength is said to be monochromatic. . blue. green. orange. indigo and violet (ROYGBIV). If we observe the visible radiations of all wavelengths.

Composition of spectrum of EMR can be represented diagramatically as follows: .

.

* a molecule may absorb energy in three ways: 1.  . when the molecule absorbs light in F. when the molecule absorb light in I.R region. what happens when radiant energy is absorbed by a molecule? * this results in gaining of energy by the molecule.I.R region.V region. 3.by increasing rotation of the molecule around the axis (rotational energy).by raising electrons to a higher energy level (transitional energy). 2.Interaction of a molecule with EMR Q. when the molecule absorbs light in visible and U.by raising the vibration of the constituent nuclei (vibrational energy).

which is characteristic for each molecule.V region.   The relative energies of transitional. vibrational and rotational are roughly in the order of 10000: 100: 1. The energy of transition can be represented by the following equation: ΔE = Es – Eg = h υ Where h is Max Planck constant and υ is the frequency of EMR at which the transition take place. it gains energy that leads to displacement of an outer electron (valence electron) and the molecule is said to be excited because electron undergoes transition from original energy level (ground state = Eg) to an excited state (Es). . When a molecule interacts with radiant energy in the visible and U.

If an excited electron returns to the ground state. may lead to bond rupture and new compounds being formed. Emission of radiant energy. This phenomenon is described as photolysis. If the excited electron returns directly to the ground state heat is evolved.V region). When excited electron returns to the ground state via second excited state. which is sufficient to exceed the energy of the formation of certain bonds. light is emitted as f1uorescence or phosphorescence (see later). it may lose absorbed energy in the form of heat.    . absorbed energy doesn't accumulate in electronic system of a molecule. In all cases.B: Absorbing large amount of energy (from far U. N. light or molecular collision.

    The relation between spectra and chemical structure The absorbance of EMR in the U. Absorbance of EMR by organic molecule is achieved by chromophoric groups (chromophores) assisted by auxochromes. where the electron of absorbing molecule gets excited. upon the number and arrangement of the electrons in organic molecule).V-VIS regions depends on structure of organic molecule (i. when it undergoes transition from the ground state to the excited state).e. What is a Chromophore? It is unsaturated organic group responsible for electronic absorption e.g. (i. .e.

g.NH2. . . changes both the λ and intensity of absorption maxima e. Notice: All auxochromes have one or more non-bonding pair of electrons.Cl.   What is an Auxochrome? It is a saturated organic group which when attached to a chromophore. it helps extending the conjugation by sharing of non-bonding electrons. -OH. . If an auxochrome is attached to a chromophore.

X or S) which don't participate in bonding. Non-bonding (n) electrons: they are of atomic ortbitals of hetero atoms (N. namely: Sigma (σ) electrons: They are bonding electrons which represent valence bonds formed due to linear overlapping of electronic clouds of S or SP orbitals. O. they are the most stable). Pi (π) electrons: they are the bonding electrons constituting the pi bonds (double bonds) and result from lateral overlap of electronic clouds of P orbitals. . n electrons occupy the highest level of ground state.e. They are of higher energy than sigma electrons.Types of electronic transition     The outer electrons in an organic molecule may occupy one of three different energy levels. They have the lowest energy level (i.

The following figure represents the different electronic energy levels and different types of transition which may occur: . while n electrons occupy either π * or σ*.     In excited state σ electrons occupy an antibonding energy level denoted as σ* and the transition is termed σ .σ * transition. π electrons occupy the antibonding π* level. The absorption spectra of organic molecules depend on the electronic transitions occurring.

They are transparent in the near UV (200-300nm).g.e. which makes of them ideal solvents for other compounds to be studied in this region. σ-Absorption:  Compounds containing only σ-electrons are the saturated hydrocarbons which absorb at < 170nm (i. O or halogens). in the far UV region). triethylamine at 199nm and chloroform at 173nm. n-electrons absorption in saturated compounds: For saturated compounds containing heteroatoms (S.    . the majority of these compounds show absorption in the near UV region e. N. Methanol at 177nm.

However. their intense absorption usually extends to the edge of the near UV region (Cut off wavelength) in the 200220nm region.  Alcohols and ethers absorb at wavelength shorter than 185nm and so they are useful as common solvents at > 200nm (that will help in quantitative analysis of drugs later). .

in order to increase sensitivity and to minimize error of the analytical method. It has characteristic shape which shows the λ of maximum absorbance (λ max). qualitative analysis). Absorption spectrum characterizes each molecule. Also λmax is used for quantitative measurement. Shape of absorption spectrum depends on electronic transitions that occurs in each organic molecule.e. therefore it is used for identification of a chemical substance (i. λ max is characteristic for each molecule. .     Absorption spectra Absorption spectrum is the plot of Absorbance (A) as a function of wavelength (λ).

B  Sometimes. absorption spectra may show a shoulder or even no absorption characteristics. .N.

.Effect of the medium (solvent).   b. a.Shifting of λmax        This feature usually happens due to change in configuration of chromophore or auxochrome groups. when two or more chromophores are present in conjugation.g.g. Types of shifting. For example.Hypsochromic shift (or blue shift) is the shift of λmax to a shorter wavelength due to removal of conjugation e. by changing polarity of the solvent.Bathochromic shift (or red shift) is the shift of λmax to a longer wavelength due to: . .General increase in conjugation e. decreasing polarity of solvent causes a red shift in n-π* transition of carbonyl compounds.g.Substitution with certain functional groups (e. -OH and NH2) .

c- Hyperchromic effect (or shift)
is increase in the intensity of absorption. It is usually induced by introduction of an auxochrome to the compound. e.g. introduction of methyl group in position 2 of pyridine increases absorption for π-π * transition.

d- Hypochromic effect (or shift)
It involves a decrease in the intensity of absorption. This is induced by introduction of groups which are able to distort the geometry of the molecule.

The presented diagram represents absorption and intensity shift.

Factors affecting absorption spectra

1- Effect of pH on absorption spectrum The spectra of compounds containing acidic (phenolic-OH) or basic (-NH2) groups are dependent on the pH of the medium. Phenol and aniline are typical examples.

 

Phenol
The U.V spectrum of phenol in acid medium is completely different from its spectrum in alkaline medium. In acid medium the benzenoid form is the predominant species; represented as phenate anion.

The spectrum in alkaline medium exhibits bathochromic shift (red shift) due to participation of the pair of electrons of oxygen in resonance with the π-electrons of aromatic ring, thus increasing the delocalization of the π -electrons, leading to the formation of conjugated system (quinonoid structure); i.e. electrons become more energetic and need less energy to be excited, therefore absorb longer λ and hyperchromic effect is observed.

while in acid medium its spectrum exhibit hypsochromic shift and hypochromic effect due to its conversion to the benzenoid species.e. Aniline Aniline behaves like phenol. its spectrum exhibits bathochromic shift and hyperchromic effect in alkaline medium due to its conversion to the quinonoid species. i.  .

Oxidation of diphenylamine will convert the benzenoid spectrum of diphenylamine to the quinonoid spectrum of the oxidised form.Effect of redox reaction on absorption spectrum. increase in conjugation leads to increase in absorbance of light by a compound.   Note: Generally. as shown in the following equation. which appears colored and exhibits hyperchromic effect. .  2.

V spectrum of known concentration of phenol as a function of pH (i.What is the isosbestic point?   On running U.e. but all spectra intersect at certain λ which is known as isosbestic point. The isosbestic point is the wavelength at which the same absorbance is given for the same concentration at different pH (i. at this point absorbance is not pH dependent but concentration dependent). . The spectrum will be shifted to different λmax by changing the pH. at different pH).e.

while  If and Ir may be canceled by means of control cuvette containing the solvent in which the substance to be anaylsed is dissolved. some is absorbed (Ia).e. reflected (Ir). i.  Therefore. I0 = Ia + It + Ir + If + Is Is = zero for clear solution. refracted (If) and scattered (Is).It  .Laws of light absorbance   When a monochromatic light having intensity (I0) is allowed to pass through absorbing medium. under experimental conditions: I0 = Ia + It Or Ia = I0 . transmitted (It).

solution) (b) at constant concentration (C) i.e.  .e. its intensity is decreased exponentially with the increase of thickness of the absorbing medium (i. log I0 / It α b or log I0 / It = K b Where K is proportionality constant.A) Bouguert -Lambert's law When a monochromatic light enters absorbing medium.

its intensity is decreased exponentially with the increase of the concentration of the absorbing medium.e log I0 / It α C or log I0 / It = KC . i. when (b) is constant.B) Beer's Law:   When a monochromatic light enters an absorbing medium.

e. l cm) and concentration is unity.e. log I0 / It α bc or log I0 / It = abc or A = abc where: A = log I0 / It = absorbance ‘a’ is a constant. (Unit of ε is L mol1. Molar absorptivity or epsilon (ε) If the unit of concentration is 1M. known as absorptivity which is the absorbance.Beer-Lambert's law:          According to lamberts' law log I0 / It α b (thickness or pathlength) According to Beers' law: log I0 / It α C (Concentration) i.cm1-) . when thickness of solution is unity (i. ‘a’ is known as molar absorptivity or epsilon (ε) or molar extinction coefficient.

lcm). . ‘a’ is known as A (1 %. When (b) is 1 cm.     A (1% . Absorptivity ‘a’. 1 cm) are characteristic for each substance and are used for qualitative purpose. A = a C or a = A/C Both ε and A (1%.1cm): If unit of concentration is 1%. can be calculated from the slope of the curve produced on plotting (A) as function of (C) at fixed (b).

g CuSO4.If the substance to be analysed is colorless.  To be measured colorimetrically.… etc. KMnO4. 2.  .The substance to be measured must be colored e.Colorimetry Principle: in colorimetry we are measuring absorbance A of ‘visible’ radiation by a colored sample under study. it must be reacted first with certain reagent (known as chromogen) to produce equivalent colored product e.g. orthophenanthrolene which reacts with ferrous (Fe2+) in buffered medium (acidic pH) to produce intense red color. organic dyes. the substance has to fit one of three categories: 1.

g. it must be converted to a certain derivative which has a suitable chromogen. in determination of ester. e.3.If the substance to be analysed is colorless and there is no suitable chromogen. which is 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.  .

we obtain straight line passing through the origin. 5. The reaction of color formation have to be rapid and quantitative. It should be stable with time. 4. on plotting A versus C at fixed b. . should obey Beer-Lambert's law. It should be unaffected by pH or the pH must be specified and maintained by suitable buffer or the measurement is carried out at λ of isosbestic point. i. to increase the sensitivity of measurement. The colored product.e. 1. What are the requirements for the colored product? It should be of intense color. 2. 3.

It should be colorless or easily separated. It should be selective (i. 3. reacts only with the substance to be analysed). The reaction to produce colored product should be of known mechanism and stoichiometric.e. 4. . 2. What are the requirements for ideal chromogen? 1. 5. Produces only one color of specified λmax. The full development of color must be rapid.

2.Instrumentation 1. It is also called ‘photo-electric colorimetric methods’. It is also called ‘visual colorimetric methods’. .Visual methods: which are applied only in case of colored samples.Photo-electric methods: which are applied in both colored and colorless samples .

matched Nessler tubes are used. Long observations weaken the eye sensitivity.  For permanent colored system colored disc. (using polychromatic light) Principle: This method is based on the comparison of the colored sample with standard series of colors. Standard colors may be freshly prepared or a permanent colored system:  For freshly prepared standards. Visual methods  Human eye is the detector in this case and its sensitivity varies with wavelength. lovibond comparator or sealed ampoules are used . Standard series method. a.I.

b) Variable depth or balancing method  Principle: can be represented as follows: According to Beer-Lambert's Law: i.e. then we calculate the concentration of the unknown sample from the equation above. we Compare a sample with a solution of known concentration of the same sample under investigation (standard solution) till the intensity of It emerging from sample and standard solution are the same. .

To apply this method we can use: 1.Duboscq-type colorimeter:  .

the depth of their immersion bring about changes in the thickness (b) of colored solutions of the sample and standard. proportional to thickness (b) and concentration of the sample (C2) is given by:  C2 = Cl (b1/b2) .  Intensity of emerging light (It) is. It depends on using a dual matched optical system made of glass plungers (p). The varying depths are measured by a vernier (V). The intensity of emerging light (It) is brought into a circular field of view (E) through prism (R).

.Hehner tube: designed to withdraw the solution from the more concentrated solution.2. until the color of the sample and that of the standard solution are matched.

.Photo-electric Instruments using monochromatic light  This method depends on the photoelectric phenomenon.II. where. the intensity of EMR is measured through the intensity of electric current produced by electrons liberated from a photosensitive metal under the influence of incident EMR.

The following figure represents schematic diagram of the instrument. Detector.       Sample compartment (cuvette). Dispersing system (or monochromator). Recorder (meter). Light source Monochromator Cuvette Detector Recorder . The instrument used consists of 5 basic components Radiant energy source (light source).

and in U.It converts polychromatic light to monochromatic light. Radiant energy source (light source)   .V range we must use deuterium lamp (D2) (or hydrogen lamp. i.) 2.In visible range.1.It must cover the desired spectral range.Monochromatic light may be obtained by one of the following systems:  .It must be of sufficient intensity . we must use tungsten lamp. .e.  . of definite range or λ. Monochromator (Dispersing system)  .

 Complementary colors can be represented by colored wheel or the following table: Colour Violet Blue Greenish blue Bluish green Green Yellowish green Yellow Orange Red Wavelength () 400-435 435-480 480-490 490-500 500-560 560-580 580-595 595-610 610-750 Complementary colour Yellowish green Yellow Orange Red Purple Violet Blue Greenish blue Bluish green .Filters  They function by selective absorption of unwanted λ and transmit the complementary color. which is needed to be absorbed by the sample to be analysed.a).

liquid and tinted glass.  .A narrower band can be obtained by using interfering filters.Notes:  . .  . .If a substance absorbs all visible light it will appear black.g. which consist of successive multiple layers of high and low refractive index material (e. MgCl2 and Agº film).Filters may be: gelatin. and if a substance doesn't absorb visible light.These types of filter transmit a wide band of 35-50 nm which is not exactly monochromatic. it will appear colorless. Interference results in a narrower band of 10-17 nm.

while in U. R O Y G B I V .V range we use prism made of quartz or fused silica.  Glass prism is used in visible range.b)-Prisms  They act by refraction of light.

 . the grating disperses the light beam into almost single λ.Through diffraction and interference.  . .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).  .c)-Grating  This one acts by diffraction and interference.Each ruled groove functions as a scattering center for light rays falling on its edge.

e.g  collimating lenses which act as condensers.V range. i.Associated optics Associated optics. glass for visible-range and quartz or fused silica for U. mirrors and diaphragms for proper alignment of the beam. Note: All optical components should be suitable for the spectral range. which helps to narrow band width. .    slit of variable width. are used to control light intensity e.

Sample compartment (Cuvette)  Cuvette is made of glass for visible range and quartz or fused silica for U. Transparent surface Obaque surface .V range. Its standard path length is 1cm (10 mm) and sometimes it is 1/2 cm.3.

4. This type requires no external source of power.photocells (Photovoltaic cell) e. N. Light falling on cell Transparent metal layer of Ago (Collecting electrode) - Photosenitive semiconductor of selenium + Metal base Plate of iron . Light Detector  There are two types of detectors: A.B. where electrons are excited and produce EMF (current) proportional to the intensity of incident light.g Barrier layer cell Principle: light falls on a semiconductor surface.

using several anodes arranged in gradually increasing potential is implemented. (greater sensitivity is obtained through magnification of EMF produced).B.B) Phototube (photomultiplier or photoemissive tube)  In order to obtain greater sensitivity when the light signal is weak.e.  N. . i. this type requires external source of power. multiplication of the initial photoelectrons by secondary emission.

. its scale is graded in absorbance or/and transmittance units. Recorder {meter}  The amplified electric signal produced in detector is fed to a sensitive galvanometer.5.

Filter photo-electric colorimeter .Commercial instruments  1.

fluctuations in source intensity of EMR source are automatically cancelled due to the two cell set up. 2. Compensating two-photocell/ colorimeter In this type. .

3. Prism spectrophotometer .

A C . and when absorbing species are affected by complexation or hydration.Deviations from Beer-Lambert's law 1) Real deviations  .This deviation decreases or disappears in very dilute solution.This happens when solute molecule in concentrated solution doesn't absorb radiant energy in the same manner as does the same molecule in dilute solution????? why ????? due to charge distribution. molecular interaction.  All of that leads to non-linear response when A is plotted against C.  .

Use of unclean optics (lenses. Irregular deviations.g finger print on the cuvette) . may result from: 1. mirrors or lamp) 2. Use of unclean handling (e. The use of unmatched cuvette (due to industrial defects) 3.2) Instrumental deviations  This type of deviation may be irregular or regular a.

B. 5. incorrect choice of filters. 2.Stray light is any radiations or λ other than that absorbed by the sample or any light that reaches the detector without passing through the sample.. Radio & T. Stray light:  .V waves also interfere. the presence of fluorescent impurities. may result from: 1. Error in slit width control (maximum opening of slit leads to non-specific λ).  . Regular deviations. Error in λ scale. hence avoid their presence close to the spectrophotometer. .b. aging of mirrors and light source.It may result from. Non-linear response of detector which gives calibration curve that doesn't pass through the origin. N. 3. Error in potentiometric reading of absorbance. 4.

 temperature effect and..3) Chemical deviations These include:  effect of pH (which leads to shifting of λmax). hydrolysis. )..  time factor (the obtained color may fade by time due to deterioration by oxidation. reduction. . etc ..

3. hence could be used for its identification.lcm).V and visible absorption spectrum usually give finger print of the sample to be analysed. Qualitative analysis. . λ max. Quantitative analysis. 1.Qualitative analysis  Absorptivity (ε or A (1% . Determination of some physical constants.Applications 1. U.. 2.

Quantitative analysis a. water.The substance to be analysed is well dissolved in a suitable solvent.  . . etc ...g. e. ether.2.Absorbance readings are taken in the expected range (e. 200-400 nm for colorless samples and 400-800 nm for colored samples). methanol.  For quantitative determination of a single component we should consider the following:  .  .The solvent is used as blank (to cancel its interference if any).g. Quantitative analysis of a single component.

Construct a calibration curve by plotting A against C at fixed b using standard series of the same chemical present in the sample.    .The absorbance of the sample to be analysed is determined under the conditions adopted during construction of the calibration curve. A Or mathematically: A = abc i. .e C = A / (a b) C .The concentration of the sample can be determined from the calibration curve.Detect λmax of the substance to be analysed after dissolving in a suitable solvent. at the characteristic λmax. . .

.b. For their quantitative analysis. X and Y must be chemically inert to each other. 3. Beer-Lambert's law must be obeyed for X and Y at their characteristic λmax.Quantitative analysis of multicomponent mixture  Consider a mixture of X and Y. 2. The absorption spectrum of X and Y should not show sever overlap. the following has to be considered: 1.

 The following 2 equations can be obtained (at b = 1cm) .

(Determination of pKa of weak acid or pkind. the following equilibrium is established:    To determine pKa.Determination of some physical constants  e. )  Consider the weak acid HB during its dissociation. Then absorbance is measured as a function of pH at λ1. .c. The pH at which the two curves are intersected represents pKa of weak acid. the absorption spectra of a fixed concentration of HB as function of pH is constructed (as shown in Figure A). (λmax of HB) and λ2 (λmax of B) (a shown in figure B).g.

.

sum up to 10). 10.Other spectrophotometric applications 1.prepare a series of dilutions of Metal ion (Mn+) ranging from 0 to 10 (i. there is no complexation reaction.  Procedure: to get the complexation ratio  . this indicates that. 0.By measuring A of these mixtures. 8 . if A of mixtures is varied.  . Determination of complexation ratio (Job's method).e... However. 0) for complexing agent (L). prepare complementary mixtures of Mn+ and L (i. .e. 9.Depending on additive property of absorbance.e. 2… 10) and ranging from 10 to 0 (i. this indicates that complexation reaction has taken place. 1. if there is no change in A.  .

. .The ratio of (Mn+ : L) can be detected by their ratio at the point of maximum or minimum absorbance (as shown in the figure).

 They may also lead to shifting of λmax.2. Effect and detection of impurities  Absorbing impurities have disastrous effects in spectrophotometric applications:  They cause distortion of the absorption curve of the mother substance leading to over estimation (i.  Accordingly it is important to detect their presence. higher absorbance).e. .

)  Construct the absorption spectra of tested sample and reference standard of the same substance contained in the tested sample. it must be equal to zero if the sample is 100% pure as the reference.I = (A'1 / A'2) – (A1/A2).   A' and A refer to the absorbance of the sample and reference standard respectively.I. . I.Detection of impurities a) Impurity index (I.

I)  S.P.P. .b) Spectrophotometric Purity Index (S.I = (A' 1/ A' 2) / (A1 / A2)  this value equals 1 in case of pure samples.

Thank You .