MULTICOMPONEN
T ANALYSIS BY UV
SPECTROSCOPY
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CONTENTS
1. Assay as a single component sample
2. Corrected interference
3. Assay after solvent extraction
4. Simultaneous equation method
5. Absorbance ratio method
6. Difference spectroscopy method
7. Derivative Spectroscopy
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ASSAY AS A SINGLE
COMPONENT SAMPLE
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CORRECTED
INTERFERENCE
If the identity, concentration and absorptivity of the absorbing
interferents are known, it is possible to calculate their contribution to the
total absorbance of a mixture.
The concentration of the absorbing component of interest is then
calculated from the corrected absorbance (total absorbance – absorbance
of interfering substance) in the usual way.
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ASSAY AFTER SOLVENT
EXTRACTION
Interfering substances can be separated from the analyte sample by
solvent extraction process.
This is mainly applied to acidic or basic drugs.
The partition coefficient of the drug in aqueous phase and organic phase
determines the extent of separation of interfering substance from the
analyte sample.
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SIMULTANEOUS EQUATION
METHOD
If a sample contains two absorbing drugs (X
and Y) each of which absorbs at the max of the
other, it may be possible to determine both
drugs by the technique of simultaneous
equations (Vierodt’s method).
The information required is:
a. The absorptivities of X at and , and
respectively.
b. The absorptivities of Y at and , and
respectively.
c. The absorbances of the diluted sample at
and , A1 and A2 respectively.
Let cx and cy be the concentrations of X and Y
respectively for diluted sample.
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SIMULTANEOUS EQUATION
METHOD
Two equations are constructed based upon the fact that at and the
absorbance of the mixture is the sum of individual absorbances of X and Y.
At , observed absorbance is
A1 = ax1 b cx + ay1 b cy … (Equation 1) [A1 = Ax1 + Ay1]
At , observed absorbance is
A2 = ax2 b cx + ay2 b cy … (Equation 2) [A2 = Ax2 + Ay2]
For measurements in 1 cm cells, b = 1
……………………………
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SIMULTANEOUS EQUATION
METHOD
Now, after rearranging Equation 2
Substituting for cy in Equation 1 and rearranging gives
And, similarly,
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Analyst can determine A1, A2, ax1, ax2 , ay1 , ay2 easily
SIMULTANEOUS EQUATION
METHOD
Criteria for obtaining maximum precision, based upon absorbance ratios,
have been suggested that place limits on the relative concentrations of the
components of the mixture.
The criteria are that the ratios should lie outside the range 0.1 - 2.0 for
the precise determination of Y and X respectively; these criteria are
satisfied only when the max of the two components are reasonably
dissimilar.
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SIMULTANEOUS EQUATION
METHOD
An additional criterion is that the two components do not interact
chemically, thereby negating the initial assumption that the total
absorbance is the sum of the individual absorbance.
The additivity of the absorbance should always be confirmed in the
development of a new application of this technique.
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ABSORBANCE RATIO
METHOD
It is a modification of the simultaneous
equation’s procedure.
It depends on the property that, for a
substance which obeys Beer’s Law at all
wavelengths, the ratio of absorbances at any
two wavelengths is a constant value
independent of concentration or path length.
For example, two different dilutions of the
same substance give the same absorbance
ratio A1/A2, i.e. 2.0. (see the figure)
In the USP, this ratio is referred to as a Q value. The British Pharmacopoeia also uses a
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ratio of absorbance at specified wavelengths in certain confirmatory tests of identity.
ABSORBANCE RATIO
InMETHOD
the quantitative assay of two
components in admixture by the
absorbance ratio method,
absorbances are measured at
two wavelengths;
one being the max of one of the
components (2)
and the other being a
wavelength of equal absorptivity
of the two components (1), i.e.,
an iso-absorptive point. (see
figure)
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ABSORBANCE RATIO
METHOD
Two equations are constructed as described above for the method of
simultaneous equation (eq. (1) and eq. (2)). Their treatment is somewhat
different, however, and uses the relationship ax1 = ay1 at (1).
Assume, b = 1 cm.
Then,
At , observed absorbance is A1 = ax1 cx + ay1 cy
At , observed absorbance is A2 = ax2 cx + ay2 cy
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𝜆1 is an isoabsorptive wavelength, and therefore, ax1 = ay1.
𝐴2 𝑎𝑥2 𝑐𝑥 + 𝑎𝑦2 𝑐𝑦
NOW, taking ratio, ∴ =
𝐴1 𝑎𝑥1 𝑐𝑥 + 𝑎𝑦1 𝑐𝑦
Dividing numerator and denominator of RHS by (cx + cy)
𝑐𝑥 𝑐𝑦
𝑎𝑥2 + 𝑎𝑦2
𝐴2 𝑐𝑥 + 𝑐𝑦 𝑐𝑥 + 𝑐𝑦
𝑤𝑒 𝑔𝑒𝑡, = 𝑐𝑥 𝑐𝑦
𝐴1 𝑎𝑥1 + 𝑎𝑦1
𝑐𝑥 + 𝑐𝑦 𝑐𝑥 + 𝑐𝑦
𝑐𝑥 𝑐𝑦
Now, let Fx=𝑐 and Fy=𝑐
𝑥 +𝑐𝑦 𝑥 +𝑐𝑦
𝐴2 𝑎𝑥2 𝐹𝑥 + 𝑎𝑦2 𝐹𝑦
𝐴𝑓𝑡𝑒𝑟 𝑠𝑢𝑏𝑠𝑡𝑖𝑡𝑢𝑡𝑖𝑛𝑔 𝑤𝑒 𝑔𝑒𝑡, =
𝐴1 𝑎𝑥1 𝐹𝑥 + 𝑎𝑦1 𝐹𝑦
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Now, please note that 𝐹𝑥 + 𝐹𝑦 = 1 𝑖. 𝑒. 𝐹𝑦 = 1 − 𝐹𝑥
𝐴2 𝑎𝑥2 𝐹𝑥 + 𝑎𝑦2 ሺ1 − 𝐹𝑥 ሻ
∴ =
𝐴1 𝑎𝑥1 𝐹𝑥 + 𝑎𝑦1 ሺ1 − 𝐹𝑥 ሻ
𝐴2 𝑎𝑥2 𝐹𝑥 + ൫𝑎𝑦2 − 𝑎𝑦2 𝐹𝑥 ൯
∴ =
𝐴1 𝑎𝑥1 𝐹𝑥 + ൫𝑎𝑦1 − 𝑎𝑦1 𝐹𝑥 ൯
𝐴2 𝑎𝑥2 𝐹𝑥 − 𝑎𝑦2 𝐹𝑥 + 𝑎𝑦2
∴ =
𝐴1 𝑎𝑥1 𝐹𝑥 − 𝑎𝑦1 𝐹𝑥 + 𝑎𝑦1
𝐴2 𝐹𝑥 ൫𝑎𝑥2 − 𝑎𝑦2 ൯+ 𝑎𝑦2
∴ =
𝐴1 𝐹𝑥 ൫𝑎𝑥1 − 𝑎𝑦1 ൯+ 𝑎𝑦1
But, 𝑎𝑥1 = 𝑎𝑦1 ; therfor, 𝑎𝑥1 − 𝑎𝑦1 = 0
𝐴2 𝐹𝑥 ൫𝑎𝑥2 − 𝑎𝑦2 ൯+ 𝑎𝑦2
∴ =
𝐴1 𝑎𝑦1 15
But, 𝑎𝑥1 = 𝑎𝑦1 ; therfor, 𝑎𝑥1 − 𝑎𝑦1 = 0
𝐴2 𝐹𝑥 ൫𝑎𝑥2 − 𝑎𝑦2 ൯+ 𝑎𝑦2
∴ =
𝐴1 𝑎𝑦1
𝐴2 𝑎𝑥2 𝑎𝑦2 𝑎𝑦2
∴ = 𝐹𝑥 − 𝐹𝑥 +
𝐴1 𝑎𝑦1 𝑎𝑦1 𝑎𝑦1
𝐴2 𝑎𝑥2 𝑎𝑦2 𝑎𝑦2
𝑖. 𝑒. = 𝐹𝑥 − 𝐹𝑥 + … ൣ∵ 𝑎𝑦1 = 𝑎𝑥1 ൧
𝐴1 𝑎𝑥1 𝑎𝑦1 𝑎𝑦1
𝐴2 𝑎 𝑥2 𝑎𝑦 2
Now, = 𝑄𝑀 ; = 𝑄𝑋 𝐴𝑛𝑑 = 𝑄𝑌
𝐴1 𝑎 𝑥1 𝑎𝑦 1
∴ 𝑄𝑀 = 𝐹𝑥 𝑄𝑋 − 𝐹𝑥 𝑄𝑌 + 𝑄𝑌
∴ 𝐹𝑥 = ሺ𝑄𝑀 − 𝑄𝑌 ሻ / (𝑄𝑋 − 𝑄𝑌 )
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𝑐𝑥 𝑄𝑀 − 𝑄𝑌
𝐹𝑥 = =
𝑐𝑥 + 𝑐𝑦 𝑄𝑋 − 𝑄𝑌
𝑄𝑀 −𝑄𝑌
After rearranging we get, 𝑐𝑥 = 𝑄 𝑋 − 𝑄𝑌
൫𝑐𝑥 + 𝑐𝑦 ൯
Now, A1 = (ax1 cx + ay1 cy) ; i.e. A1 = (ax1 cx + ax1 cy);
∴ ൫𝑐𝑥 + 𝑐𝑦 ൯= A1/ax1
𝑄𝑀 − 𝑄𝑌 𝐴1
∴ 𝑐𝑥 = ൬ ൰
𝑄𝑋 − 𝑄𝑌 𝑎𝑥1
Similarly, we can derive
𝑄𝑀 − 𝑄𝑋 𝐴1
∴ 𝑐𝑦 = ቆ ቇ
𝑄𝑌 − 𝑄𝑋 𝑎𝑦1
𝐴2 𝑎𝑥 2 𝑎𝑦2
=𝑄 𝑀 ; =𝑄 𝑋 𝐴𝑛𝑑 =𝑄 𝑌
𝐴1 𝑎 𝑥1 𝑎𝑦1
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Analyst can determine A1, A2, ax1, ax2 , ay1 , ay2 easily
DIFFERENCE
SPECTROSCOPY METHOD
The selectivity and accuracy of spectrophotometric analysis of samples
containing absorbing interferants may be markedly improved by the
technique of difference spectrophotometry.
The essential feature of a difference spectrophotometric assay is that the
measured value is the difference absorbance (ΔA) between two equimolar
solutions of the analyte in different chemical forms which exhibit different
spectral characteristics.
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DIFFERENCE
SPECTROSCOPY METHOD
The criteria for applying difference spectrophotometry to the assay of a
substance in the presence of other absorbing substances are that:
a. reproducible changes may be induced in the spectrum
of the analyte by the addition of one or more reagents
b. the absorbance of the interfering substances is not altered by
the reagents.
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DIFFERENCE
SPECTROSCOPY METHOD
The simplest and most commonly employed technique for altering the
spectral properties of the analyte is the adjustment of the pH by means
of aqueous solutions of acid, alkali or buffers.
The ultraviolet-visible absorption spectra of many substances containing
ionisable functional groups, e.g. phenols, aromatic carboxylic acids and
amines, are dependent on the state of ionization of the functional groups
and consequently on the pH of the solution.
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DIFFERENCE
SPECTROSCOPY METHOD
The absorption
spectra of
equimolar
solutions of
Phenylephrine, a
phenolic
sympathomimeti
c agent, in both
0.1M
hydrochloric
acid (pH 1) and
0.1M sodium
hydroxide (pH
13) are shown in 21
figure.
DIFFERENCE
SPECTROSCOPY METHOD
The ionization of the phenolic group in alkaline solution generates an
additional n (non-bonded) electron that interacts with the with the ring π
electrons to produce a bathochromic shift of the λmax from 271nm in acidic
solution to 291 nm and an increase in absorbance at the λ max (hyperchromic
effect).
The difference absorption spectrum is a plot of the difference in absorbance
between the solution at pH Such wavelengths of equal absorptivity of the
two species are called isobestic or iso-absorptive points.
Above 278 nm the alkaline solution absorbs more intensely than the acidic
solution and the ΔA is therefore positive. Between 257 and 278 nm it has a
negative value.
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DIFFERENCE
SPECTROSCOPY
The measure value in a
METHOD
quantitative difference
spectrophotometric assay is
the ΔA at any suitable
wavelength measured to the
baseline, e.g. ΔA1 at λ1 or
amplitude between an
adjacent maximum and
minimum, e.g. ΔA1 at λ2 and
λ1.
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DIFFERENCE
SPECTROSCOPY METHOD
At λ1, ΔA = Aalk – Aacid
Where Aalk and Aacid are the individual absorbances in 0.1M sodium
hydroxide and 0.1M hydrochloric acid solution respectively.
(If the individual absorbance, and are proportional to the concentration of the
analyte and path length, the also obeys the Beer – Lambert’s law and a modified
equation may be derived.)
Where ΔA is the difference absorptivity of the substance at the wavelength of
measurement.
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DIFFERENCE
SPECTROSCOPY METHOD
If one or more other absorbing substances are present in the sample
which at the analytical wavelength has identical absorbance in the
alkaline and acidic solutions, its interference in the spectrophotometric
measurement is eliminated.
The selectivity of the ΔA procedure depends on the correct choice of the
pH values to induce the spectral change of the analyte without altering
the absorbance of the interfering components of the sample.
The use of 0.1M sodium hydrochloric acid to induce the ΔA of the
analyte is convenient and satisfactory when the irrelevant absorption
arises from pH intensive substances.
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DERIVATIVE SPECTROSCOPY
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DERIVATIVE SPECTROSCOPY
First derivative spectrum of an absorption band is characterized by a
maximum, a minimum and cross over point at the λmax of the
absorptionband.
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DERIVATIVE SPECTROSCOPY
Advantages of First derivative spectroscopy:
(1) Precise determination of the λmax can
be obtained from the zero crossing of the
first derivative.
(2) Improved spectral resolution
(3) Discrimination of broad bands
Resolution enhancement in derivative
spectroscopy
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DERIVATIVE SPECTROSCOPY
Second derivative spectrum is characterised by two satelite
maxima and an inverted band of which the minimum
corresponds to the λmax of the fundamental band.
Second derivative spectrum eliminates the broad band
absorption.
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DERIVATIVE SPECTROSCOPY
Derivative spectra may be generated by three techniques.
[Link] of the optical system
Spectrophotometers with dual monochromators, photo detectors used–
wavelength selected with difference of 1-3nm.
Generates a signal with an amplitude proportional to the slope of the
spectrum over the wavelength interval.
Disadvantage: Expensive ,Restricted to the recording of first derivative
spectra only.
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DERIVATIVE SPECTROSCOPY
2. To generate derivative spectra -- electronic differentiation of the
spectrophotometer analogue signal.
Resistance capacitance (RC) modules are highly dependent on
instrumental parameters, the scan speed and the time constant.
Standard solution of analyte is employed to calibrate the measured value
under the instrumental condition selected
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DERIVATIVE SPECTROSCOPY
[Link] upon microcomputers differentiation
Micro computers incorporated in to or interfaced with
spectrophotometer may be programmed To provide
Derivative spectra during or after scan
To measure derivative amplitudes between specified wavelengths
To calculate concentrations and associated statistics from the measured
amplitudes
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