1

MULTICOMPONENT ANALYSIS
BY UV SPECTROSCOPY

FInal. Y. Dr. Archana Naik

 Types of Quantitative Analysis
by UV-VISIBLE spectroscopy
2
Single component analysis Multiple component
(Sample Contains only one Analyte ) analysis (Sample Contains
more than one Analyte )

1.Use of Standard absorptivity 1. Assay as a single component

value
2. Use of calibration graph
3. Single point standardization
4. Double point standardization
analysis
2. Assay using absorbance
corrected for interference
3. Simultaneous equation method
4. Derivative spectrophotometric
method
5. Difference spectrophotometry
6. Absorbance ratio method(Q-
Absorbance method)
 Multicomponent analysis: Available techniques
3
 Simultaneous estimation of drug combination is generally
done by separation using chromatographic methods like
HPLC, GC and HPTLC etc.
 These methods are accurate and precise with good
reproducibility, but the cost of analysis is quite high owing
to expensive instrumentation, reagent and expertise.
 Hence UV-VIS spectrophotometer is used to develop simpler
and cost effective method for simultaneous estimation of
drugs for routine analysis of formulations.
 Spectrophotometric analysis fulfils such requirement where
the simultaneous estimation of the drug combination can be
done with similar effectiveness as that of chromatographic
 Why Multicomponent analysis
4

 Many excipients in formulation interfere with analyte of

interest during analysis which makes the single component
analysis very difficult.
 The pharmaceutical analyst frequently encounters the situation
where the concentration of one or more substances is
required in samples known to contain other absorbing
substances, which potentially interfere in the assay.
 A number of modifications to the simple spectrophotometric
procedure are available to the analyst, which may eliminate
certain sources of interference and permit the accurate
determination of all of the absorbing components.
 Those techniques are called as multicomponent analysis.
 INTRODUCTION
5

 Each modification of the basic procedure may be

applied if certain criteria are satisfied.
 Correct choice of procedure for particular sample is
very much essential for accurate result.
 The basis of all the spectrophotometric techniques for
multicomponent samples is the property of analyte at all
wavelengths:
 the absorbance of a solution is the sum of absorbance of the
individual components; or
 the measured absorbance is the difference between the total
absorbance of the solution in the sample cell and that of the
solution in the reference cell.
 Multicomponent analysis
6

 Assay as a single component analysis

 Assay using absorbance corrected for interference
 Simultaneous equation method
 Derivative spectrophotometric method
 Difference spectrophotometry
 Absorbance ratio method(Q-Absorbance method)
 Geometric correction method
 Chemical Derivatization
 1. Assay as a single component analysis

 The concentration of component in the sample which contain

absorbing substances can be analyzed by single component
analysis method if interfering agent (other component) have a
sufficiently small absorbance at the wavelength of
measurement.
 A systematic error of less than 1% is acceptable. Eg. If the
contribution to a total absorbance of 1.00 from interfering agent is
less than 0.01, and if there is no chemical reaction between the
components, then sample can be analysed by simple direct
measurement of absorbance at its λ-max.
 Eg. Pediatric Paracetamol Elixir
 At large dilution (Appro. 3250 times) of the sample the
 2. Assay using absorbance corrected for interference
8

 If the identity, concentration and the

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 minus absorbance
of the interfering subs).
 Assay using absorbance corrected for interference
9

 The λ-max of ephedrine hydrochloride and chlorocresol are

257 nm and 279nm resp. and A1% 1cm values in 0.1 M HCl
solution are
 Ephedrine hydrochloride at 257nm = 9.0
 Ephedrine hydrochloride at 279 nm =0
 Chlorocresol at 257nm= 20.0
 Chlorocresol at 279 nm =105.0
 Calculate conc of ephedrine hydrochloride and chlorocresol
in a batch of ephedrine hydrochloride injection, diluted 1
ml to 25 ml with water.
 A at 279nm = 0.424 A at 257nm =0.972 b=1cm
 Assay using absorbance corrected for interference
10

 A) Since ephedrine does not absorb at 279 nm, calculate the conc of
chlorocresol from the A 279 of the diluted inj.
0.424=105 x 1x C
C= 0.00404g/100ml
 Therefore conc of chlorocresol in inj = 0.00404 X 25
= 0.1010gm/100ml = 1.010mg/ml
 B) Calculate conc of chlorocresol at 257 nm in diluted inj.
A= 20 X 1X 0.00404 =0.081
 C) Calc. conc. Of ephedrine hydrochloride from corrected absorbance at
257 nm
 Corrected absorbance at 257 nm= 0.972-0.081= 0.891
 0.891= 9.0 X1 XC= 0.0990 gm /100ml
 Therefore conc of ephedrine hydrochloride in inj = 0.0990 X 25
 3. Assay after solvent extraction of the sample
11

 If the interference from the other absorbing substances is

large or if its contribution to the total absorbance can not be
calculated, it may be possible to separate the absorbing
interferents from the analyte by solvent extraction procedures.
 Solvent extraction method is best for acidic or basic drugs
whose state of ionization determines their solvent
partitioning behavior.
 The selection of proper pH of aqueous medium and
immiscible solvent(organic solvents) may effectively
separate the interferents from the analyte.
 The concentration of which may be obtained by taking
absorbance of the extract containing analyte.
Sulfamethoxazole and trimethoprim combination tablet.

12
 Uses: cotrimazole is antibiotic used forbladder infections.[1] Other uses include
for middle ear infections and travelers' diarrhea. S
 Sulfamethoxazole contains amino groups which is responsible for absorbance
at around 265 nm.
 But absorption is interfered by presence of trimethoprim which contain
pyridine ring and also absorb at 265 nm. Hence it is difficult to carry out
determination in combination tablet. Various methods are available to
overcome this problem.
 One approach is separation of two components from each other
completely and then measuring absorbance of each component separately.
 If the interference from other substance is large, if its contribution to total
absorbance can’t be calculated, it may be done by separating the absorbing
interferent from each other by solvent extraction. This is particularly
appropriate for acidic and basic drugs whose state of ionization
determines the portioning behavior.
13

 Absorption spectra of (a) trimethoprim, (b) sulfamethoxazole and (c) mixtures

of sulfamethoxazole and trimethoprim (Concentration of each components is
6.0 µg mL-1 )
 assay of sulfamethoxazole and trimethoprim tablet
14
 In the assay of sulfamethoxazole and trimethoprim tablet, both
drugs absorb at λ max of each other. Sulfamethoxazole is
acidic in nature.(bacteriostatic sulfonamide
antibiotic :Amphoteric sulfonamides behave as weak acids
and are much more soluble in alkaline) It dissolve in base to
form sodium salt.
 Trimethoprim is basic in nature so remains unionized in
NaOH. The unionized moiety can be extracted using
CHCl3 because it is a non-polar solvent.
 Based on the partition coefficient most of the trimethoprim
will go into CHCl3 but some will remain in aqueous phase.
This process is repeated several times so that all trimethoprim
will be extracted.
 Assay of sulfamethoxazole and Trimethoprim
tablet
15

 Now organic solvent contain almost all

trimethoprim with negligible
sulfamethoxazole and aqueous alkaline phase
contains almost all sulfamethoxazole with
negligible amount of trimethoprim.
 Thus both the drugs can now be determined as

individual entities without interference by

other.
 4. Simultaneous Equation method
16

 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. (Vierodts Method)
 •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
respectively.
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.

17
 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)

Substract equation 4 from equation 3

A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1

A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1)

Cy = (A1 aX2 - A2 aX1) / (aX2 aY1 - aX1aY2)

18
18
 At λ1 A1 = aX1* Cx + aY1* Cy (1)

At λ2 A2 = aX2* Cx + aY2* Cy (2)

•Multiply the equation (1) with aY2 and (2) with aY1

A1 aY2 = aX1 Cx aY2 + aY1 Cy aY2 (6)

A2 aY1 = aX2 Cx aY1+ aY2 Cy aY1 (7)

Subtract equation 6 from equation 7

A2 aY1 - A1 aY2 = aX2 Cx aY1 - aX1 Cx aY2

A2 aY1 - A1 aY2 = Cx (aX2 aY1 - aX1aY2 )

Cx = (A2 aY1 - A1 aY2 ) / (aX2 aY1 - aX1aY2 ) (8)

19
19
 Cy = (A1 aX2 - A2 aX1) / (aX2 aY1 - aX1aY2)

Cx = (A2 aY1 - A1 aY2 ) / (aX2 aY1 - aX1aY2 )

•These equations are known as simultaneous equations and by
solving these simultaneous equations we can determine the
concentration of X and Y in the sample.
•Ex. Sodium benzoate and caffeine

20
 Examples:

21

 Estimation of Losartan potassium and Hydrochlorthiazide in tablets.

 Estimation of Salbutamol and Theophylline from tablets.
 Estimation of Amlodipine besylate and Enalepril maleate from tablets.
 Estimation of Ibuprofen and Pseudoephedrine hydrochloride from
tablets.
 Estimation of Salbutamol and Theophylline from tablets
 5. Difference spectroscopy
22

 Difference spectroscopy provides a sensitive method for

detecting small changes in the environment of a chromophore or
it can be used to demonstrate ionization of a chromophore
leading to identification and quantitation of various components
in a mixture.
 The essential feature of a difference spectrophotometric assay is
that the measured value is the difference absorbance (Δ A)
between two equimolar solutions of the analyte in different
forms which exhibit different spectral characteristics.
23
 The criteria for applying differential spectroscopy in assay of
substance in presence of other absorbing substance are:
 Reproducible changes may be induced in spectrum of
analyte by addition of one or more reagents
 The absorbance of interfering substance is not altered by
the reagents
 Simplest and most commonly employed technique for altering
spectral properties analyte is adjustment of pH by means of
aqueous solution of acid, alkalis or buffers.
 The absortion spectra of many substances containing ionisable
functional groups eg. Phenols, carboxylic acid and amines are
dependent on state of ionization of the functional group and
consequently pH of the solution.
 Assay of phenylephrine injection
24

 The absorption of spectra of equimolecular solution of

phenylephrine, a phenolic sympathomimetic agent, in both 0.1
N HCl(pH-1) and 0.1 N NaOH (pH-13)
 The ionisation of phenolic group in alkaline solution generate
additional n-electrons (non banded) that interact with π electron
to produce bathochromic shift of λmax from 271nm in acidic
solution to 291 nm and increase in absorbance of λmax
(Hyperchromic shift)
 The difference absorption spectrum is a plot of the difference in
absorbance between the solution at pH 13 and pH 1 against
wavelength. It may be generated automatically using double
beam recording spectrophotometer with the solution at pH 13 in
the sample cell and the solution at pH 1 in the reference cell.
25

 At 257 and 278 nm both solutions have identical

absorbance and consequently exhibit zero difference
in absorbance. Such wavelength of equal
absorptivity of the two species are called isobestic
or isoabsorptive points.
 Above 278 nm the alkaline solution absorb more

intensity than acidic solution and ΔA value is

therefore positive.
 Between 257nm and 278nm it has a negative value.
26

 At λ1, ΔA is Aalkaline-A acidic

 Where Aalk and Aacid are the individual
absorbances at λ1 in 0.1 M NaOH and 0.1 m HCl
respectively.
 If individual absorbances Aalk and Aacid are
proporational to the concentration of the analyte and
pathlength , the ΔA also obeys beers lamberts law
and modified equation may be derived ΔA= Δabc
 Where Δa is the difference in absorptivity of
substances at wavelength of measurement.
 A B
A) The Spectrum of compound in B(acid) and A(Base)
27 B) The difference spectrum of B relative to A
27
6. 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.
28
29
 At λ1 A1 = aX1* Cx + aY1* Cy (1)
At λ2 A2 = aX2* Cx + aY2* Cy (2)
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)
(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]
30
 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)

31
Cy= {(QM-Qx)/(Qy-Qx)}* ( A1/ay1)
 Examples:

32

 Estimation of Rifampicin and Isoniazide in pharmaceutical

dosage forms.
 Estimation of Spiranolactone and hydroflumethiazide.
 Estimation of Nalidixic acid and Metronidazole from tablets.
 Estimation of Noscapine, Chlorpheniramine Maleate and
Ephedrine hydrochloride from tablets.
 7. Derivative spectroscopy
 Derivative spectroscopy uses first or higher derivatives of
33 absorbance with respect to wavelength for qualitative

analysis and for quantification.

 For the purpose of spectral analysis in order to relate

chemical structure to electronic transitions, and for

analytical situations in which mixture contribute
interfering absorption, a method of manipulating the
spectral data is called derivative spectroscopy.
 Derivative spectrophotometry involves the conversions of a

normal spectrum to its first, second or higher derivative

spectrum. (As shown in figure 1). In the context of
derivative spectrophotometry, the normal absorption
spectrum is referred to as the fundamental, zero order, or D 0
34
35
Zero order
spectrum
36

First order
spectrum

Second order
spectrum
 Derivative spectroscopy -D1 spectrum
37
 The first derivative D1 spectrum is a plot of the rate
of change of absorbance with wavelength against
wavelength i.e. a plot of the slope of the
fundamental spectrum against wavelength or a plot
of dA/dλ vs. λ.
 The maximum positive (λ1 & λ2) and maximum

negative (λ4 & λ5) slope respectively in the D0

spectrum correspond with a maximum and a
minimum respectively in the D1 spectrum.
 The λmax (λ3) in D0 spectrum is a wavelength of

zero slope and gives dA/dλ = 0 in the D1 spectrum.

 Derivative spectroscopy-D2 spectrum
38
 The second derivative D2 spectrum is a plot of the curvature of
the D spectrum against wavelength or a plot of d 2A/ dλ2 vs. λ.
 The maximum absorbance at λ2 &λ4 (Max. absorbance with

λ) in the D1 spectrum gives a minimum in the D 2

spectrum, and the maximum slope at λ1 &λ5 in the D
spectrum gives two small maxima called satellite bands in
the D2 spectrum.
 The wavelength of maximum slope and zero curvature in the

D spectrum correspond with cross-over points in the D 2

spectrum.
 It also shows two additional positive satellite bands either

side of the main band.

 Derivative spectroscopy
39
 Derivative spectroscopy-Significance
40

 These spectral transformations confer two

advantages on derivative spectrophotometry.
 First order spectrum is of narrower spectral

bandwidth than its fundamental spectrum.

 A derivative spectrum therefore shows better

resolution of overlapping bands than the

fundamental spectrum and may permit the
accurate determination of the λmax of the
individual bands.

Quantification by Derivative spectroscopy
41

 If we assume that the zero-order spectrum obeys

Beer’s law, there is a similar linear relationship
 between concentration and amplitude for all orders
of derivative:
 Zero order A= εb c
 First order
λ wavelength
A absorbance
ε extinction coefficient
 nth order b sample path length
c sample concentration
 Derivative spectroscopy
42

 For single component quantification the selection of

wavelengths for derivative spectra is not as simple as
for absorbance spectra because there are both
positive and negative peaks.
 Obtaining derivative spectra Derivative spectra can be
obtained by optical, electronic, or mathematical methods.
 Optical and electronic techniques were used on early
UV-Visible spectrophotometers but have largely been
superseded by mathematical techniques.
Determination of piroxicam and its major metabolite
5-hydroxypiroxicam in human plasma by zero-
crossing
first-derivative
43 spectrophotometry.
 Piroxicam is readily absorbed after oral administration and
It is extensively metabolised by hepatic cytochrome P450
enzyme, principally to the hydroxyl metabolite.
 The zero-order UV spectra of solutions of 5.0 mg/ ml
piroxicam, 5.0 mg/ml 5-HP and their mixture (1:1) in a
mixture of 1.0 M HCl-acetonitrile (1:1, v:v) over the
wavelength range 200-400nm is shown in fig 1 and
wavelength range 280–380 nm is shown in fig 2 .
 Due to extensive overlap of the spectral bands,
conventional UV spectrophotometry cannot be used for
the quantification of both substances in the presence of each
44

Piroxicam 5-hydroxypiroxicam
 zero-order UV spectra
45
Fig.1.Absorption(zero-order)UV
spectra of 5.0 mg ml1 piroxicam
(——),
5.0 mg ml1 5-HP (- - -), and
their mixture in the ratio 1:1 (···), in
acetonitrile.
Mixture Fig2. Absorption(zero-order)
343.5 -5HP UVspectra of 5.0 mg ml1 piroxicam
(——),
5.0 mg ml1 5-HP (- - -), and
332.5 -P
their mixture in the ratio 1:1 (···),
in a mixture of 1.0 M HCl-acetonitrile
 Zero Crossing first order derivative
spectrophotometry
46

 Zero-crossing first order derivative

spectrophotometry permits a more selective
identification and determination of the two
compounds in a mixture.
 The zero-crossing method, involves measurements

absorbance of piroxicam at an wavelength which is

zero crossing point of 5-hydroxypiroxicam.
 Analysis of 5-hydroxypiroxicam is carried out at

wavelength which is zero-crossing point for Analyte

piroxicam.
 first-order derivative
47

 The first-order derivative spectra of these samples

were recorded over the wavelength range 325–360
nm against a blank of a mixture of 1.0 M HCl-
acetonitrile (1:1, v:v).
Fig 3 (a) showed that the
Signals at 332.5 nm, D1(332.5), (zero-crossing
wavelength point of piroxicam) are proportional to
the concentration of 5-HP.
Signals at 343.5 nm, D1(343.5), (zero-crossing
wavelength point of 5-HP) are proportional to the
concentration of piroxicam.
48

 Therefore analysis of piroxicam was carried out at

D1343.5 nm, and analysis of 5-hydroxypiroxicam
at D1332.5 nm.
 Fig. 3(b) shows a typical set of the first-order

derivative spectras of mixture containing P and 5-

HP in1.0 M HCl-acetonitrile (1:1, v:v), at
concentrations 0.5, 1.0, 3.0, 6.0 and 10.0 µg/ml.

..\mph\UV multicomponent analysis case study\klopas

-PIROXICAME derv spectroscopy bioanalysis.pdf
49

D1(332.5) -zero-crossing
wavelength point of
piroxicam

D1(332.5) -zero-crossing
wavelength point of 5-
Hydroxypiroxicam

 Fig. 3. (a) First-order derivative spectra of 5.0 mg ml1 piroxicam (···),

5.0 mg ml1 5-HP (- - -), and their mixture in ratio 1:1 (——), in a
mixture of 1.0 M HCl-acetonitrile (1:1, v:v);
 (b) First-order derivative spectra of spiked plasma extracts containing
mixture of piroxicam and 5-HP in ratio 1:1 at concentrations 0.5, 1.0,
3.0, 6.0 and 10.0 mg ml
 References

•A. H. Beckett and J. B. Stenlake Practical

Pharmaceutical Chemistry, Part I and Part II, 4th Edition
•Vogel Text Book of Quantitative Chemical Analysis,
Longman, London
•Principles of instrumental analysis, D.A. Skoog and leary
J.J. Saunders college publishing , New york.
•Instrumental methods of Analysis by Gary D Christian –
6th edition
•Determination of piroxicam and its major metabolite
5-hydroxypiroxicam in human plasma by zero-crossing
first-derivative spectrophotometry, A. Klopas, I. Panderi,
M. Parissi-Poulou , Journal of Pharmaceutical and
Biomedical Analysis 17 (1998) 515–524.

50
• Enlist a method for quantitative analysis of 2
drugs which absorb at wavelength maxima of
each other.
•Enlist a method for quantitative analysis in
which analysis is carried out at isobestic point
and wavelength maxima.
1. Isoabsorptive point
2. Name a multicomponent analysis technique
based on recording absorbances of sample
in two different mediums/solvents.
3. Enlist a method for analysis of multicomponent
formulation using zero cross
over wavelength
51
4 marks
1.Explain derivative spectroscopic method for
UV analysis
2.Enlist various methods of multicomponent
analysis using UV spectrophotometric technique.
3.Explain a suitable technique for quantitative
analysis of 2 drugs which absorb at wavelength
maxima of each other.

4.Enlist various methods of multicomponent

analysis using UV-spectrophotometry. Explain
any one in detail.
5.Explain multicomponent analysis by UV
52
spectroscopy using Simultaneous equation
method.