Application of UV Visible spectroscopy in pharmaceuticals

Pharmaceutical Analysis

1. Application of UV-Visible
Spectroscopy

2. 1. Qualitative Analysis:
The UV spectra of most compounds are of limited value for
qualitative analysis as compared to IR and Mass spectra. Qualitative
analytical use of UV spectra has largely involved λ-max and absorptivities,
occasionally includes absorption minima. In pharmacopoeias, absorption
ratios have found use in identity tests, and are referred to as Q-values in
USP.

2. Quantitative Analysis:
UV spectroscopy is perhaps the most widely used spectroscopic
techniques for the quantitative analysis of chemical substances as pure
materials and as components of dosage forms.

3. A)    Single component Analysis:
Direct Analysis: Essentially all compounds containing conjugated double bond
or aromatic rings, and many inorganic species absorb light in the UV-visible regions. In
these techniques the substance to be determined is dissolved in suitable solvent and diluted
to the required concentration by appropriate dilutions and absorbance is measured.
Such as Paracetamol, Metformin, Diazepam, Atenolol etc.
Indirect Analysis: (Analysis after addition of some reagent) indirect methods
are based on the conversion of the analyte by a chemical reagent that has different spectral
properties. Chemical derivatization may be adopted for any of the several reasons. 1) If the
analyte absorbs weakly in the UV region. 2) The interference form irrelevant absorption
may be avoided by converting the analyte to a derivative, which absorbs in the visible
region, where irrelevant absorption is negligible. 3) This technique can be used to improve
the selectivity of the assay in presence of other UV radiation absorbing substance. 4) Cost.
Various inorganic compounds can be analysed using complexing reagents such as a)
Thiocyanate can be used for the analysis of iron, cobalt, molybdenum b) Iodide ion used
for analysis of bismuth, palladium c) Organic chelating agents are also used for the
analysis of many cations eg. phenanthroline used for the determination of iron, dimethyl
glycoside used for analysis of nickel and diphenyldithiocarbazone used for analysis of lead
Various organic compounds can be analysed by chemical reagent such as a) NEDA dye
used for analysis of sulphacetamide b) Ferric chloride is used for analysis of aspirin

4. Methods of calculating concentration in single component analysis
•        By using the relationship: A = a b c
•        By using the formula: Cu = (Au/As) X Cs
•        By using the equations: Y = mX + C
•        By using the Beer’s curve
 
B) Multi component Analysis:
a) Simultaneous Equations method:
If a sample contains two absorbing drugs (X and Y) each of which
absorbs at the λ-max of the other (λ1 and λ2), it may be possible to
determine both the drugs by the simultaneous equations method.
Criteria for obtaining maximum precision, below mentioned ratio
should lie out side the range 0.1-2.0
(A2/A1) / (aX2/aX1) and (aY2/aY1) / (A2/A1)

5. The information required is
•        The absorptivities of X at λ1 and λ2, aX1 and aX2
•        The absorptivities of Y at λ1 and λ2, aY1 and aY2
•        The absorbances of the diluted sample at λ1 and λ2, A1 and A2

Let Cx and Cy be the concentration of X and Y respectively in the sample
The absorbance of the mixture is the sum of the individual absorbances of
X and Y

At λ1 A1 = aX1* Cx + aY1* Cy (1)
At λ2 A2 = aX2* Cx + aY2* Cy (2)
Multiply the equation (1) with aX2 and (2) with aX1

A1 aX2 = aX1 Cx aX2 + aY1 Cy aX2 (3)
A2 aX1 = aX2 Cx aX1+ aY2 Cy aX1 (4)

6. A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1
A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1)
Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) (5)

Same way we can derive
Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1) (6)

Equations 5 and 6 are known as simultaneous equations and by solving
these simultaneous equations we can determine the concentration of X and
Y in the sample.

7. b) Q-Absorbance ratio method
The absorbance ratio method is a modification of the
simultaneous equations procedure. It depends on the property that, for a
substance, which obeys Beer’s law at all wavelength, the ratio of
absorbances at any two wavelengths is a constant value independent of
concentration or path length.
In the quantitative assay of two components in admixture by the
absorbance ratio method, absorbances are measured at two wavelengths,
one being the λ-max of one of the components (λ2) and other being a
wavelength of equal absorptivity of two components (λ1), i.e. an iso-
absorptive point.
At λ1 A1 = aX1* Cx + aY1* Cy (1)
At λ2 A2 = aX2* Cx + aY2* Cy (2)

8. Now divide (2) with (1)
A2/A1 = (aX2* Cx + aY2* Cy)
(aX1* Cx + aY1* Cy)

Divide each term with (Cx + Cy)
A2/A1 = (aX2* Cx + aY2* Cy) / (Cx + Cy)
(Cx + Cy) (aX1* Cx + aY1* Cy) / (Cx + Cy)

Put Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy)
A2/A1 = [aX2 Fx + aY2 Fy] / [aX1 Fx + aY1Fy]
Where Fx is the fraction of X and Fy is the fraction of Y i.e. Fy = 1-Fx
There fore A2/A1 = [aX2 Fx + aY2 (1-Fx)] / [aX1 Fx + aY1(1-Fx)]
= [aX2 Fx + aY2 – aY2Fx] / [aX1 Fx + aY1 – aY1Fx]

9. At iso-absorptive point aX1 = aY1 and Cx = Cy
There fore A2/A1 = [aX2 Fx + aY2 – aY2Fx] / aX1
= (aX2 Fx/ aX1) + (aY2/ aX1) –( aY2Fx/ aX1)
Let Qx = aX2/aX1 , Qy = aY2/aY1 and absorption ratio Qm = A2/A1
Qm = Fx Qx + Qy - Fx Qy
= Fx (Qx-Qy) + Qy
Fx = (Qm – Qy) / (Qx – Qy)
(3)
From the equations (1)
A1 = aX1 (Cx + Cy) there fore Cx + Cy = A1 / aX1
There fore Cx = (A1/aX1) – Cy (4)

From the equation (3)
Cx / (Cx + Cy) = (Qm – Qy) / (Qx – Qy)
There fore Cx / (A1 / aX1) = (Qm – Qy) / (Qx – Qy)
There fore Cx = [(Qm – Qy) / (Qx – Qy)] X (A1 / aX1) (5)

10. c)  Derivative spectroscopy

11. Derivative spectroscopy involves the conversion of a normal spectra to its
first, second or higher derivative spectra.
The normal spectrum is known as fundamental, zero order or D
0 spectra.
The first derivative spectrum (D
1) is a plot of the rate of change of
absorbance with wavelength against wavelength, i.e. plot of ΔA/Δλ vs. λ.

The second derivative spectrum is a plot of Δ
2A/ Δλ
2 vs. λ. For the
quantitative estimation of binary mixtures by the derivative spectroscopy,
first of all we have to find out the Zero Crossing Points (ZCP) for both the
components (A and B). Now select ZCP for A and B so that at that
particular ZCP other component shows remarkable absorbance. Now
prepare calibration curve of A at the ZCP of B and of B at the ZCP of A.

Find out the unknown concentration using calibration curves.

12. 3) Determination of Dissociation constant of an indicators
Indicators give different color at different pH. Methyl red is red in color in acidic
medium and is yellow in alkaline medium because in acidic medium it remains as HMR
(Unionized form) and in alkaline medium as MR
- (Ionized form).
HMR MR
- + H
+
(Red) (Yellow)
Ka = [(MR
-) (H
+)] / (HMR)
Therefore pKa = pH – Log [(MR
-) (H
+)]

4)  Determination of composition of Metal ligand Complex.
M + L = Complex
Metal ligand complexes absorb radiation in the UV-Visible region.
Different ligands which are used are I
-, Br
-, Cl
-, OH
-, NH3, ethylene diamine, CN
-
The high absorptivity of some complexes is due to formation of charge transfer complex
molecule. Charge transfer complex usually forms between metal ion and ligand in which
metal ion acts as the electron acceptor while ligand is electron donor.
There is two methods for the determination of composition of complex first is Mole ratio
method. In this technique concentration of one of the components of the complex is kept
constant and other is increased and the absorbance of the resulting solution is measured.
Now from the plot of absorbance Vs concentration. Another method is Job’s curve method
(Continuous variation method).
5) As a detector in HPLC

13. 6) Detection of conjugation
It helps to show relationship between different groups particularly with respect to
conjugation.
Conjugation may be carbon-carbon double bond or carbon-oxygen double bond.
It gives idea about the number and locations of substituent attached to the carbon
of conjugated system.
7) Detection of Geometrical isomers
Trans-isomers exhibit λmax at slightly longer wavelengths and have larger
extinction co-efficient than the cis-isomer.
Example: trans-stilbens λmax: 294 → ℇ = 24000
cis-stilbens λmax: 278 → ℇ = 9350
8) Detection of impurity
UV absorption spectroscopy is one of the best methods for detecting impurity in
organic compounds.
Example: The common impurity in cyclohexane is benzene. It can be easily
detected by its absorption at 255 nm.

14. 9) Tautomeric equilibrium
UV-Visible Spectroscopy can be used to determine the percentages of
various keto and enol forms present in tautomeric equillibrium.
10) Analysis of mixtures of compound
There are various spectrophotometric methods available that can be used
for the analysis of mixtures. Such as analysis of Combiflam tablet
(Paracetamol and Ibuprofen) by simultaneous equation method.
11) Photometric titrations
It is used for the location of the equivalent point in the titration if analyse,
reagent or product absorbs radiation.
A Photometric titration curve is a plot of absorbance as a function of the
volume of titrant. The equivalent point is the intersection point in the two
linear regions. One linear region appears as the titration is started and the
second linear region appears after equivalence point.

15. THANK YOU