Syllabus: SPECTROPHOTOMETRY Principle of … Principle of spectrophotometry ... a particular...

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Dr. K. SIVAKUMARDepartment of Chemistry

SCSVMV [email protected]

SPECTROPHOTOMETRY

Course: M.Sc (Chemistry) Analytical Chemistry Unit: III

Syllabus:

Principle of spectrophotometry

Types of spectrophotometer

Applications - Dissociation constants of an indicator

simultaneous spectrophotometric determinations

Determination of Stoichiometry of Complexes

Job’s method of continuous variation

mole ratio and slope ratio analysis

Advantages and limitations photometric titrations

Colorimetry, standard series method

duplication method - balancing method

photoelectric colorimeter.

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Electromagnetic Waves - Terminologies

Electromagnetic wave parameters:

Wavelength ( λ): Wavelength is the distance between the consecutive peaks or crests

ν

Wavelength is expressed in nanometers (nm)1nm = 10 -9 meters = 1/1000000000 meters1A°°°° = 10-10 meters = 1/10000000000 meters

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ν

Frequency ( νννν): Frequency is the number of waves passing through any point pe r second.

Frequency is expressed in Hertz (Hz)

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Wave number ( ): Wave number is the number of waves per cm.

ν

ν

1=

c

ννλ

=Where,

λλλλ is wave length

is wave number

νννν is frequency

c is velocity of light in vacuum. i.e., 3 x 108 m/s

ν

Wavelength, Wave number and Frequency are interrela ted as,

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UVX-rays IRγγγγ-rays RadioMicrowaveVisible

nm

EM waves

10-4 to 10 -2 10-2 to 100 100 to 102 102 to 103 103 to 105 105 to 107 107 to 109

Electromagnetic Spectral regions

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Electromagnetic radiation sources

EM radiation Spectral method Radiation source

Gamma rays Gamma spec. gamma-emitting nuclides

X-rays X-ray spec.Synchrotron Radiation Source (SRS), Betatron (cyclotron)

UltravioletUV spec.

Hydrogen discharge lamp

VisibleVisible spec.

tungsten filament lamp

InfraredIR spec.

rare-earth oxides rod

MicrowaveESR spec.

klystron valve

Radio waveNMR spec.

magnet of stable field strength

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Electromagnetic Spectrum – Type of radiation and Energy change involved

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Electromagnetic Spectrum – Type of radiation and Energy change involved

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Lambert

fraction of the monochromatic light absorbed by a homogeneous medium is independent of the intensity of the

incident light and each successive unit layer absorbs an equal fraction of the light incident on it

Lambert’s law

Beer’s law

Beer

fraction of the incident light absorbed is proportional to the number of the absorbing molecules in the light-path and will increase with increasing concentration or sample thickness.

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Beer–Lambert law / Beer–Lambert– Bouguer law / Lambert – Beer law

log (I0/I) = εεεε c l = AWhere, I0 - the intensity of incident lightI - the intensity of transmitted lightε - molar absorptivity / molar extinction coefficient in cm2 mol-1 or L mol-1 cm-1.c - concentration in mol L-1

l - path length in cmA - absorbance (unitless)

Molar absorptivity

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Absorption intensity εεεε

wavelength of light corresponding to maximum absorption is designated as λλλλmax and can be read directly from the horizontal axis of the spectrum

Absorbance (A) is the vertical axis of the spectrum A = log (I 0/I)I0 - intensity of the incident light; I - intensity of t ransmitted light

λλλλmax

Intensity of absorption is directly proportional to the transition probability

A fully allowed transition will have εεεεmax > 10000 A low transition probability will have εεεεmax < 1000

εεεεmax

εεεεmax = 20000

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Applications of Beer-Lambert’s Law

In determining the concentration of solutions absorbing in UV or visible region.

For Example, if we have a standard solution (i.e., a solution with known concentration) then,

let consider that,

s

s

A – absorbance of standard solution

C – concentration of standard solution

s sA C l=∈

According to Beer-Lambert’s law,

s

s

A

Cl= ∈

or

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Applications of Beer-Lambert’s Law…

Now, to find out the concentration of a test solution (i.e., solution with unknown concentration),

we can measure the absorbance of test solution and according to Beer-Lambert’s law,

u

u

A

Cl=∈

u

u

Where, A – absorbance of test solution

C – concentration of test solution

Comparing equations u

u

A

Cl=∈s

s

A

Cl= ∈ &

uu s

s

AC C

A= ×

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Deviations from Beer’s Law & / Limitations of Beer- Lambert’s Law

�deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostaticinteractions between molecules in close proximity

often assumed that Beer’s Law isalways a linear plot describing therelationship between absorbanceand concentration

Beer’s Law successfully describes thebehaviour of dilute solutions only. Athigh concentrations (ie greater than 10-

2 M) there is interaction betweenabsorbing particles such that theabsorption characteristics of the analyteare affected.

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� scattering of light due to particulates in the sample

� fluoresecence or phosphorescence of the sample

� non-monochromatic radiation, deviations can be minimized by using a relatively flat part ofthe absorption spectrum such as the maximum of an absorption band

�by shifts in the position of a chemical or physical equilibrium involving the absorbing species.

Deviations from Beer’s Law & / Limitations of Beer- Lambert’s Law…

Schematic of a single-beam spectrophotometer


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Schematic of a double-beam spectrophotometer


Schematic of a split-beam spectrophotometer


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Light Sources Light Sources Light Sources Light Sources

UV Spectrophotometer

• Hydrogen Gas Lamp

• Mercury Lamp

Visible Spectrophotometer

• Tungsten Lamp


Dispersion Devices

• Non-linear dispersion

• Temperature sensitive

• Linear Dispersion

• Different orders

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Photomultiplier Tube Detector

Anode

• High sensitivity at

low light levels

• Cathode material

determines spectral sensitivity

• Good signal/noise

• Shock sensitive

The Photodiode Detector

• Wide dynamic range

• Very good

signal/noise at

high

light levels

• Solid-state device

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Cells Cells Cells Cells

• UV Spectrophotometer - Quartz (crystalline silica)

• Visible Spectrophotometer - Glass

•

Open-topped rectangular standard cell (a)

and apertured cell (b) for limited sample volume

Cell Types I

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Cell Types II

Micro cell (a) for very small volumes and flow-through cell (b) for automated applications

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Beer–Lambert law / Beer–Lambert– Bouguer law / Lambert – Beer law

Bouguer

Actually investigated therange of absorption Vsthickness of medium

Lambert

Extended the conceptsdeveloped by Bouguer

Beer

Applied Lambert’sconcept to solutions ofdifferent concentrations

Bernard

?Beer released the resultsof Lambert’s concept justprior to those of Bernard

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Spectrometer

The instrument used for recording the absorption spectra of a compound is calledspectrometer.

The different components present in various types of spectrometer are shown here.

UV - hydrogen discharge lamp

Visible - tungsten filament lamp

IR - electrically heated rod of rare-earth oxides

Microwave - klystron valve

NMR - magnet of stable field strength.

Variable slit, rheostat, etc

Prism,ilter,monochromator,grating

Cuvette, test-tube,

cell

Photographic plate, photocell,

photomultiplier, photoconductivity

device etc

Galvanometer; pen recorder; cathode ray oscillograph

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Colorimetric analysis

Principle: Colorimetry analysis method is useful in determining the concentration ofcoloured solutions using the visible region (400nm–750nm) of electromagneticspectrum and Beer Lambert’s law.

If the test solution is colourless then a suitable complexing agent can be added to testsolution to get coloured which will absorb light.

Example: For cuprous ions (Cu2+) estimation NH4OH can be added to get blue colour.

Instrumentation: Tungsten filament lamp is used to generate visible region (400nm – 750nm) light.

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Colorimetric analysis…

The molecules in the cuvette absorb light and the remaining light istransmitted to the photocell. In photocell,

Current generated Amount of light transmittedα

But the amount of light transmitted depends on the depth of colour oftest solution. i.e., concentration of test solution.

If high concentration solution is analysed then, more number ofmolecules will be in the path of light and more amount of light will beabsorbed.

So, the amount of light transmitted will be very less and generates onlyless current.

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Colorimetric analysis: Applications

useful in estimating the concentration of coloured solutions

Example: Estimation of CuSO4 by colorimetry

Series of CuSO4 solution with known concentration are prepared andammonium hydroxide is added to each solution to get blue colour.

Absorbance of each standard CuSO4 solution is measured with same filterand tabulated.

Concentration (C) of CuSO4 Absorbance (A)

0.0001 A1

0.0002 A2

0.0003 .

0.0004 .

0.0005 .

0.0006 A6

test solution At

A= C l∈

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UV Visible Spectroscopy

Principle:

Visible and ultraviolet spectroscopy is a study of electronic spectra of organicmolecules which are found in the wavelength region of 100nm-400nm (UV region)and 400nm-750nm (Visible region).

UV and visible radiations absorbed by the molecules will bring transition of outer shellelectrons(σ, π and n electrons).

According to molecular orbital theory when a organic molecule absorbs UV or visibleradiations its electrons are promoted from a bonding to an antibonding orbital.

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The Ultraviolet region [10 – 800nm]

The Ultraviolet region may be divided as follows,

1. Far (or Vacuum) Ultraviolet region [10 – 200 nm]

2. Near (or Quartz) Ultraviolet region [200 – 380 nm]

3. Visible region [380 - 800 nm]

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• In UV - Visible Spectroscopy , the sample is irradiated with the broad spectrum of the UV - Visible radiation

• If a particular electronic transition matches the energy of a certain band of UV - Visible, it will be absorbed

• The remaining UV - Visible light passes through the sample and is observed

• From this residual radiation a spectrum is obtained with “gaps” at these discrete energies – this is called an absorption spectrum

UV - VISIBLE SPECTROSCOPY

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Instrumentation

sam

ple

refe

renc

e

dete

ctor

I0

I0 I2

I1

log(I0/I) = A

200 700λλλλ, nm

monochromator/beam splitter optics

UV-VIS sources

I

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Radiation source, monochromator and detector

Two sources are required to scan the entire UV-VIS band:Deuterium lamp – covers the UV – 200-330Tungsten lamp – covers 330-700

The lamps illuminate the entire band of UV or visible light; the monochromator (grating or prism) gradually changes the small bands of radiation sent to the beam splitter

The beam splitter sends a separate band to a cell containing the sample solution and a reference solution

The detector (Photomultiplier, photoelectric cells) measures the difference between the transmitted light through the sample (I) vs. the incident light (I0) and sends this information to the recorder

Instrumentation…

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Virtually all UV spectra are recorded solution-phase

Only quartz is transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra 380 – 800nm

Concentration: 0.1 to 100mg10-5 to 10-2 molar concentration may safely be used

Percentage of light transmitted: 20% to 65%At high concentrations, amount of light transmitted is low, increasing the

possibility of error

A typical sample cell (commonly called a cuvet):Cells can be made of plastic, glass or quartz(standard cells are typically 1 cm in path length)

Sample Handling

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Structure identification

• Useful in the identification of a newly synthesized compound.

The spectrum of unknown compound can be compared with the absorption spectrum ofseveral known compounds in the literature.

On comparison, If the spectrum of unknown compound correlates with a specificallyknown compound then the structures of both will also be similar. This method is calledfinger printing technique.

SPECTROPHOTOMETRY - Applications

Concentration of impurities

During the purification process of a compound, if the absorption spectrum is recorded ata particular interval of time the decrease in the concentration of impurities can bemonitored. The purification can be continued till it gives a less value of absorbance.

To study the rate of a reaction

To study the rate of formation of product in a reaction, the absorbance of product can bemeasured at definite intervals of time. We know that, the absorbance is directlyproportional to concentration and hence the absorbance value will be increasing withrespect to time.

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SPECTROPHOTOMETRY – Applications – Determination of D issociation constant

Note the several curve cruse to same point is called isobestic point, it is veryimportant because two champers has same absorptivities, since at this point (pH)has not effect.

Example: Absorption spectrum of 3.7×10 -4 M methyl red as a function of pH between pH 4.5 and 7.1

If the absorbance with pH plots at λmax, we get S-shape curve as shown in figure bellow:

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SPECTROPHOTOMETRY – Applications – Determination of D issociation constant of Indicator

Methyl Red

two forms of methyl red absorb strongly in the visible range, the ratio (MR-)/(HMR) may be determined spectrophotometrically

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SPECTROPHOTOMETRY – Applications – Determination of D issociation constant of Indicator….

The composition of a mixture of HMRand MR- may be calculated fromabsorbancies A1 and A2 at wavelengthsλ1 and λ2 using, at unit cell thickness

Absorbance of acid and basic form of methyl red at two wavelengths

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SPECTROPHOTOMETRY – Applications – Determination of D issociation constant of Indicator….

From the plots of absorbancy versus wavelength which just obtained, twowavelengths are selected for analyzing mixtures of the acidic and basic forms ofmethyl red.

Absorbancy of HMR and MR-as a function of wavelength λ.

pK can be calculated by

knowing the ratio of (MR-)/(HMR)

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• Mixtures:Determining the concentration of mixtures the components of which absorb in the same spectral regions is possible.

• Strategy of the analysis. Total absorption at some wavelength of a two component mixture: Atotal,λ1 = AΜ,λ1 + AN,λ1.

• Each should obey Beer's law at this wavelength as long as concentration is sufficiently low. The contribution from each would then be:

• AM,λ1 = εM,λ1bCM and AN,λ1 = εN,λ1bCN. and • Atotal,λ1 = εM,λ1bCM + εN,λ1bCN. • Similarly at some other wavelength we would

have, • Atotal,λ2 = εM,λ2bCM + εN,λ2bCN. • .εb can be determined for each using standard

solutions.• Take absorbance readings of mixture at the two

λs.• Substitute into above so that there are two

equations with two unknowns.

Simultaneous analysis of mixture

The total absorbance of a solution at any given wavelength is equal to the sum of the absorbances of the individual components in the solution.

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Two cases for analysis of a mixture.

(a) Spectra of the pure components have substantial overlap.

(b) Regions exist in which each component makes the major contribution.

Visible spectrum of MnO4– , Cr2O72– , and an unknown

mixture containing both ions.

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Methods for obtaining the stoichiometry of complex

(determination of the composition of complexes)

M + nL = MLnContinuous-variation method

(Job’s method)

Methods for obtaining the stoichiometry of complex

(determination of the composition of complexes)

M + nL = MLn1) Continuous-variation method

(Job’s method)

This method is based on the measure-ment of a series of solutions in whichmolar concentrations of two reactants varybut their sum remains constant. Theabsorbance of each solution is measuredat a suitable wavelength and plottedversus the mole fraction of one reactant. Amaximum in absorbance occurs at themole ratio corresponding to the combiningratio of the reactants.

Continuous variations plots for 1:3, 1:2 and 1:1 metal to ligand complexes.

Plot peak

0.5 for 1:1 (MX) complex

0.33 for 1:1 (MX2) complex

0.67 for 2:1 (M2X) complex

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(Job’s method)

Continuous variations plot for

1:1 metal to ligand complexe.

Plot peak

0.5 for 1:1 (MX) complex

0.33 for 1:1 (MX2) complex

0.67 for 2:1 (M2X) complex

2) Mole-ratio method

A series of solution is preparedin which the analyticalconcentration of one reactant isheld constant while that of otheris varied. A plot of absorbanceversus mole ratio of thereactants is then prepared. Ifthe reaction is sufficientlycomplete, two straight lines ofdifferent slopes are obtained.The intersection of theextrapolated lines correspondsto the combining ratio in thecomplex.

Unlike the method of continuousvariations, the measuredabsorbance does not have to becorrected by subtracting theabsorbance.

Mole-ratio plots for 1:1 and 1:2 metal-to-ligand complexes.

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3) Slope-ratio method

This method, used mainly in studying weak complexes, requires that the formationreaction can be forced to completion with a large excess of either metal or ligand. Twosets of solutions are prepared : The first contains various amounts of metal ion each withthe same large excess of ligand, while the second consists of various amounts of ligandeach with the same large excess of metal. For the reaction

xM + yL = MxLy

when L is present in large excess, the concentration of product formed is limited by theconcentration of the metal, or

[MxLy] = CM / x

If Beer’s law obtains,

A = εb[MxLy] = εbCM / x

and a plot of A versus CM will yield a straight line with a slope of εb/x.

Similarly, for the solutions containing M in large excess,

[MxLy] = CL / y A = εb[MxLy] = εbCL / y

The ratio of the two slopes is the combining ratio for the reaction

(εbCM / x )(εbCL / y) = x / y

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Colorimetry – Balancing Method – Duboscq colorimeter

•Unknown and standard solutions are taken incylinders.

•The transparent plungers are moved up anddown until the colours seen from the top of eachcylinder become identical.

•From the readings of the depth of the samples,the concentration of unknown can be evaluatedusing the equation given below,

uu s

s

AC C

A= ×

Jules Duboscq

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Colorimetry – Balancing Method – Duboscq colorimeter

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Colorimetry – Multiple Standard Method

• Unknown solution is taken in 50ml or 100ml Nessler tube and made up to the mark

• The solution is compared with a series of standard solutions with known concentrations

• The concentration of unknown solution will then be equal to that of the known solution whosecolour it matches exactly.

• If the unknown matches with the solution containing 0.3g and 0.4g, a series of 0.32g, 0.34g,0.36g…. Containing solutions are prepared to get an exact or nearly exact match

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Colorimetry – Duplication Method

• Unknown solution is taken in a Nessler tube and appropriate colour developing reagent isadded to produce a colour.

• In another Nessler tube the colour developing reagent is taken and made up little lower thanthe mark.

• From a burette, a standard solution is added to this Nessler tube with constant agitation untilthe colour of unknown solution becomes duplicated in this job.

• This method is less accurate

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Photoelectric Colorimeters

Single beam (Evelyn) photon-electric colorimeter – Gurdeep Chatwal – pp.2.216

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Photoelectric Colorimeters

Double beam photon-electric colorimeter – Gurdeep Chatwal – pp.2.217

Typical photometric titration curves. Molar absorptivities of analyte titrated, product, and the titrant are εA, εP, εT, respectively.

Photometric titrations

Absorbance of solution is

measured after adding titrant

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(a) Spectrophotometric titration of 30.0 ml of EDTA in acetate buffer with CuSO4 in the same buffer.

Upper curve: [EDTA] = [Cu2+] = 5.00 mM. Lower curve: [EDTA] = [Cu2+] = 2.50 mM.

(b) Trans formation of data to mole fraction format.

Photometric titrations

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Dr. K. SIVAKUMARDepartment of Chemistry

SCSVMV [email protected]

Good Luck!

Syllabus: SPECTROPHOTOMETRY Principle of … Principle of spectrophotometry ... a particular...

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