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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Electromagnetic Waves - Terminologies
Electromagnetic wave parameters:
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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Electromagnetic Waves - Terminologies
Electromagnetic wave parameters:
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
Types of spectrophotometer
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Schematic of a double-beam spectrophotometer
Types of spectrophotometer
Schematic of a split-beam spectrophotometer
Types of spectrophotometer
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Light Sources Light Sources Light Sources Light Sources
UV Spectrophotometer
• Hydrogen Gas Lamp
• Mercury Lamp
Visible Spectrophotometer
• Tungsten Lamp
Types of spectrophotometer
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
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SPECTROPHOTOMETRY – Applications – Determination of D issociation constant
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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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