Study Guide
Molecular Spectroscopy UV Spectroscopy-2
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University:
Kentucky State University -
Course:
CHE 414 | Instrumental Analysis Academic year:
2024
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Views:
264
Pages:
26
Author:
drvnomyhv
Molecular spectroscopy
UV Spectroscopy-2 Beer’s Lambert Law
Molecular absorption spectroscopy is based on the
measurement of the transmittance T or the absorbance
A of solutions contained in a transparent cells having a
path length of b
Transmittance, T = I/ I0
% Transmittance, %T = I/ I0 x100 %
Absorbance (A) = - log T
A = log I / T
A = log I0 /I = εbc
A = εbc Beer’s Lambert Law
The measurement of light absorption by a solution of
molecules is governed by the Beer–Lambert Law Beer’s Lambert Law
Absorbance (A) = - log T
A = log I0 /I = εbc
A = εbc
ε: Molar Absorption Coefficient, Molar Excitation
Coefficient and Molar Absorptivity.
Measuremant of how strongly a chemical species
absorbs light at a given wavelength. It is intrinsic
property of the species.
Ranges from 0 to 100,000 L/mol.cm
c: Concentration (mol/L, g%....)
b: pathlength of the cell in cm, usually 1 cm (cm) Beer’s Lambert Law
Absorbance (A) = - log T
A = log I0 /I = εbc
A = εbc
where Io is the intensity of incident radiation; It is the intensity of
transmitted radiation
A (1%, 1 cm) is the absorbance of a 1% w/v (1 g/100 ml) solution in
a 1 cm cell; b is the pathlength in cm (usually 1 cm); and c is the
concentration of the sample in g/100 ml What are the concentrations of the following solution of
Carbimazole in g/100 ml and mg/100 ml?
A (1%, 1 cm) value= 557 at 291 nm, measured
absorbance=0.557 at 291 nm.
0.001 gm%, 1 mg%
Question 1: A protein with an Excitation
Coefficient E1%= 16 yields an absorbance of
0.73 when measured in a 0.5 cm cell. Calculate
the weight concentration.
Solution:
A =E1%bc
c= A/ E1%b
= 0.73/(16*0.5)
= 0.091 g% (g/100ml) Question 2: Cytosine has a molar absorbtivity of 6*103 at
270 nm at pH7. Calculate the absorbance and percent
transmission of 1*10-4 and 1*10-3 M cytosine solution in
1mm cell.
Solution:
For 1*10-4 M solution:
Abs= 6*103 x 0.1 x 1*10-4
= 0.06
T%:
Absorbance (A) = - log T
A = log I / T
T%= 10-0.06 x100%= 87.10%
Solution (Continued)
For 1*10-3 M solution:
Abs= 6*103 x 0.1 x 1*10-3
= 0.6
T%:
Absorbance (A) = - log T
A = log I / T
T%= 10-0.6 x100%= 25.12% Question 3: 100 ml of a solution contains 0.050 g
of a coloured compound that absorbs exactly
half the incident light intensity at 500 nm when
the solution is in a 1.00 cm wide
spectrophotometer cell. The cell width that would
absorb 75% of the incident light intensity is Limitation of Beer-Lambert Law
The linearity of the Beer lambert law is limited by
chemical and instrumental factors. Causes of
nonlinearity include: UV-Vis Spectroscopy
Strengths
An easy-to-use, cheap and robust method offering good precision
for making quantitative measurements of drugs in formulations.
Routine method for determining some of the physico-chemical
properties of drugs, which need to be known for the purposes of
formulation.
Some of the problems of the basic method can be solved by the use
of derivative spectra.
Limitations
Only moderately selective. The selectivity of the method depends on
the chromophore of the individual drugs, e.g a coloured drug with an
extended chromophore is more distinctive than a drug with a simple
benzene ring chromophore.
Not readily applicable to the analysis of mixtures. Applications of UV spectroscopy in
pharmaceutical analysis
A robust, workhorse method for the quantification of drugs in
formulations where there is no interference from excipients.
Determination of the pKa values of some drugs.
Determination of partition coefficients and solubilities of drugs.
Used to determine the release of drugs from formulations with
time, e.g. in dissolution testing.
Can be used to monitor the reaction kinetics of drug
degradation.
The UV spectrum of a drug is often used as one of a number
of pharmacopoeial identity checks. Assay Examples
1)
2)
3)
4)
5)
Tablet powder containing 0.25g of frusemide is shaken with
300 ml of 0.1M NaOH to extract the acidic frusemide.
The extract is then made up to 500 ml NaOH (solution A)
A portion of the extract is filtered and 5 ml of the filtrate is made
to 250 ml with 0.1N NaOH (solution B)
The absorbance of the diluted extract is measured at 271 nm
The A(1%, 1 cm) value at 270 is 580
Calculate the % of the stated content in a sample of frusemide
tablets if
Stated content per tablet: 40 mg of frusemide.
Weight of 20 tablets:1.657 g
Weight of tablet powder taken for assay: 0.5195 g
Absorbance reading: 0.596 % of the stated content = Practical Wt/Theoratical Wt * 100%
= Practical Wt/0.25 * 100%
Abs = A(1%)*b*c
c= 0.596/580*1
c= 1.0276*10-3 g% (solution B, diluted)
Dilution Factor: 250/5=50
Concentration (solution A, Concentrated) = c *DF
= 1.0276*10-3 x 50 = 0.0514 g%
I need amount
0.0514 gm frusemide
100 ml solution
????
500 ml
0.2569 gm frusemide is actually present in powder taken
% of the stated content= 0.2569/0.25 *100% = 102.76% Chloramphenicol eye drops assay
Dilute a volume containing 25mg of chloramphenicol to
250 ml with water. dilute 10 mL to 100 mL and measure
absorbance of the resulting solution at the maximum at
278 nm.
Calculate the content of Chloramphenicl taking 297 as
the value of A(1%,1cm) at the maximum at 278 nm.
Absorbance=(0.114)
% Content of Chloramphenicol Spectrophotometric Mixture Analysis Atotal= A1 +A2…..
AbsλA= Abs AλA + Abs BλA
= εA,λA.b.cA + εB,λA.b.cB
AbsλB= Abs AλB + Abs BλB
= εA,λB.b.cA + εB,λB.b.cB Question
Concentration A Concentration B
(mol/L)
(mol/L)
Abs. at 510 nm
Abs. at 575 nm
1.5*10-1
0.0
0.714
0.097
0.0
Unknown
6*10-2
Unknown
0.298
0.671
0.757
0.330
1) Calculate the four molar absorptivities (εA(510), εA(575),
εB(510) , εB(575)
2) Calculate the molarities of the two salts A and B in the
Unkown. Solution
1) ε = Abs/bc
εA(510) = 4.76
εA(575) = 0.647
εB(510) = 4.967
εB(575) = 12.617
So,
0.671=4.76 CA+ 4.967 CB
0.330= 0.647 CA + 12.617 CB
2) CAunknown = 0.12 M
CBunknown = 0.02 M Types of UV-Visible Instruments
(Spectrophotometers)
All of these instruments (Single, double
beam) have:
- A light source
- A filter or monochromator
- A sample holder
- A detector.
Single Beam Spectrophotometer
Double Beam Spectrophotometer Double Beam Spectrophotometer
The double beam instrument has a single source and a
monochromator and then there is a splitter and a series
of mirrors to get the beam to a reference sample and the
sample to be analyzed, this allows for more accurate
readings Single vs Bouble Beam Instruments
In choosing a solvent, consideration must be
given not only to its transparency, but also to its
possible effect on absorbing system.
Next table represents a list of some common
solvents and the approximate wavelength below
which they cannot be used because of
absorption (cutoff wavelength)
Solvent
Lower λ limit,
(nm)
Water
180
Ethanol
220
n-Hexane
200
Cyclohexane
200
Diethyl ether
210
Acetone
330
Molecular Spectroscopy UV Spectroscopy-2
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