Multicomponent Analysis

by using
UV visible Spectroscopy
p
py

Fixed Dose Combinations

Fixed-dose combinations ((FDCs)) are also known as combination p
products
are combinations of two or more active drugs produced in a single dosage form.
List of some fixed dose combinations available in indian market
1 Clonidine and Hydrochlorothiazide

Antihypertensive

2 Clonidine and Chlorthalidone

Antihypertensive

3 Tizanidine, Nimesulide and Paracetamol

NSAID

4 Tizanidine and Mefanamic acid

NSAID

5 INH and Ethambutol and Rifampicin

Anti Tuberculosis

6 Zidovudine and Lamivudine and Navirpine

Antiviral

7 Stavudine and Lamivudine and Navirpine

Antiviral

8 M
Metformin
tf
i and
d Glibenclamide
Glib
l id

A
Antidiabetics
tidi b ti

9 Metformin and Glimepride

Antidiabetics

Slide 01

Antihypertensive drugs

Captopril

1) ACE inhibitor & Diuretics

50%

50%

Hydrochlorothiazide + Captopril

90%
Hypertensive population

A i t
Angiotensinogen
i

Hypertensive
yp
p
population
p

Renin Proteolytic enzyme

Angiotensine
goe s eI

Little
eb
biological
o og ca ac
activity
y

(Decapeptide)

Angiotensin converting
enzyme
Stimulates the release of
another hypertensive agent
(aldosterone).

Angiotensine II

Vasoconstrictor

(Octapeptide)

The ultimate result of ACE action is hypertension (increased BP)

Inhibition of ACE would decrease blood pressure

therefore it is important target for design of antihypertensive agent
Slide 02

2) ACE inhibitor & Calcium channel blockers

The angiotensin-converting enzyme (ACE) inhibitor benazepril plus the

calcium-channel blocker Amlodipine
p

The angiotensin-converting
Th
i t
i
ti enzyme (ACE) inhibitor
i hibit Enalapril
E l
il plus
l th
the
calcium-channel blocker felodipine

These drug classes have different spectra of side effects, none of which are
additive.
The combination
Th
bi ti
produces
d
the
th
same antihypertensive
tih
t
i
effect
ff t as hi
higher
h
doses of either constituent, the exposure to side effects is reduced and the
therapeutic ratio is increased.

Slide 03

The therapeutic ratio can

be increased
The therapeutic index (also known as therapeutic ratio), is a comparison of
the amount of a therapeutic agent that causes the therapeutic effect to the
amountt that
th t causes death
d th
Potentiation

Potentiation is the synergistic effect

on drug A by adding a dose of drug
B without a therapeutic effect.
An example is the combination of
Bisoprolol or Enalapril with a low
dose of Hydrochlorothiazide it self
without direct antihypertensive effect
but beneficial
in
controlling
h
hypertension
t
i
d
due tto it
its di
diuretic
ti
effect.

Cancellation

Cancellation is a phenomenon in
which the adverse effects of one
drug are nullified by the addition of a
second
An example is the hypokalemic
effects of thiazide diuretics are
counteracted
by
the
slight
hyperkalemic effect of an ACEi hibit )
inhibitor).
Slide 04

Combination therapy of two (or more) different classes of drugs with a

common therapeutic effect
Amoxicillin and Clavulanate
Clavulanate
Acts by inhibiting bacterial degradation of Amoxicillin
Amoxicillin, rather than having any direct
therapeutic activity itself
Carbidopa and Levodopa
Carbidopa itself has no beneficial therapeutic effect, but it inhibits the systemic
decarboxylation and inactivation of levodopa before it crosses the bloodbrain
barrier and is converted to dopamine, the active metabolite that relieves the
symptoms of Parkinsons disease.

Slide 05

Advantages of Fixed Dose Combination Products

1)) Increase in the therapeutic
p
ratio
2) Reduced administration costs stem from simplified packaging, fewer
prescriptions, and fewer dispensing fees
3) Reducing the number of pills diminishes the complexity of the regimen, so
that improved patient adherence is expected with combination products.
4) Combination products make particular sense in the treatment of infectious
disease, where partial adherence can lead to the development of drugresistant strains and a threat to public

Slide 06

Disadvantages of Fixed Dose Combination Products

1) These fixed dose combinations can lead to polypharmacy
2) Dose of one ingredient cannot be altered
3) Different pharmacokinetic properties can pose difficulty in frequency of
administration and in case of development of an Adverse Drug Reaction
(ADR)
4) It is difficult to withdraw the suspected drug alone
5)) A combination makes it more difficult to p
pinpoint
p
the offending
g agent
g
responsible for the adverse reaction.
6) Some FDCs when combined lead to increased toxicity. For instance, the
anti-TB drugs,
g , Streptomycin,
p
y , Kanamycin
y
and Capremycin
p
y
cannot be
combined, as they have the same side effects (oto and nephro-toxicity)

Slide 07

S
Spectrophotometric
t
h t
t i techniques
t h i

Simultaneous estimation of drug

g combination is g
generally
y done by
y separation
p
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 it is
worthwhile to

develop simpler and cost effective method for simultaneous

estimation of drugs for routine analysis of formulation

formulation. Spectrophotometric
analysis fulfils such requirement where the simultaneous estimation of the drug
combination can be done with similar effectiveness as that of chromatographic
methods.

Slide 08

There are various spectrophotometric methods available which can be used

for the analysis of combination samples.
Following methods can be used
1) Simultaneous equations method (Vierodts method)
2) Absorbance
Ab b
ratio
ti method
th d (Q
(Q-Absorbance
Ab b
method)
th d)
3) Derivative spectrophotometric method
4) Geometric correction method
5) Orthogonal polynomial method
6) Difference spectrophotometric method
7) Chemical derivatisation method

Slide 09

Need of Multicomponent Analysis

The spectrophotometric assay of drugs rarely involves the measurement of
absorbance of sample containing only one absorbing component.
The pharmaceutical analyst frequently encounters the situation where concentration
of one or more substances is required to be determined in presence of other
absorbing
b bi substances
b t
which
hi h potentially
t ti ll iinterfere
t f
i th
in
the assay.
Example:
1

Rx

Rx

Rx

Brimonidine Tartarate

Olmisartan Midoxomil

Ezitamibe

Timolol Maleate

Hydrochlorothiazide

Simvastatin
Slide 10

If the recipe of sample formulation is available to the analyst. The analyst can
identify concentration of substance interfering and its extent of interference.
If the unknown interference arise due to manufacturing impurities, decomposition
products and formulation excipients, if not removed imparts a systematic error to the
analysis of drug in sample.
A number of modifications to the simple spectrophotometric procedure used for
determination single component are available, which may eliminate certain sources
off interference
f
and permit the accurate determination off one or all off the absorbing
components.
The basis of all the spectrophotometric technique for multicomponent analysis is the
property
t that
th t att allll wavelengths
l
th
a) The absorbance of a solution is the sum of the absorbance of the individual
components or
b) The measured absorbance is the difference between the total absorbance
between the total absorbance of the solution in the sample cell and that of the
solution in the reference (blank) cell.

Slide 11

Analysis of Caffeine , Metamizol,

Acetaminophen in pharmaceutical
formulation

Absorption spectra of Caffeine (a), Metamizol (b)

Acetaminophen (c), and their mixtures (d)

Slide 12

Simultaneous equation method (Vierodts method)

This method is advantageous over other methods, because it is simple and
involves less complicated calculations.
If sample contains two absorbing drugs (X and Y) each of which absorb at the max
of the other, it may be possible to determine both the drugs by the technique of
simultaneous
i lt
equations.
ti

Absorbance

For further calculation following information is used

Y

Mixture

Wavelength

a) The absorptivities of X at 1 & 2, are ax1 and ax2

b) The absorptivities of Y at 1 & 2, are ayy1 and ayy2
c) The absobances of diluted sample at 1 & 2 are
A1 and A2
Let Cx and Cy be the concentration (gm/100ml)
of X and Y respectively in the diluted sample.

Slide 13

Two equations are based upon the fact that at 1 and 2 the absorbance of the
mixture is the sum of the individual absorbance of X and Y.

At 1

A1 = ax1 bcx + ay1 b cY

(1)

At 2

A2 = ax2 bcx + ay2 b cY

(2)

For measurement in 1 cm cell, b=1.

Rearranging equation. (2)

A2 ax2 cx
ay2
Substituting for CY in eq. (1) and rearranging gives
Cy =

Cx =

Cy =

A2 ay1 A1ay2

(3)

ax2 ay1 ax1ay2

A1 ax2 A2ax1
ax2 ay1 ax1ay2

(4)

Slide 14

Criteria for obtaining maximum precision by using simultaneous equation

Following ratio should lie outside the range 0.1 2.0 for the precise determination
of Y and X respectively.
A2 / A1
ax2 / ax1

and

ay2 / ay1
A2 / A1

The criteria are satisfied only when the max of the two component are
reasonably dissimilar.
In addition to above there is one more criteria that compound do not react
chemically, which will prove initial assumption wrong that the total absorbance is
the sum of individual absorbances.
In British Pharmacopoeia the assay of quinine-related alkaloids and Cinchonine
related alkaloids are based on this technique.
Slide 15

Assay of caffeine and sodium benzoate injection by the simultaneous

equation method
The composition of injection is

Anhydrous
A
h d
caffeine..125
ff i
125 mg
Sodium benzoate ..125 mg
Water to
. 1 ml

Dilute injection,100 times

Anhydrous caffeine..125 mg
Sodium benzoate ..125
125 mg
Water to
. 100 ml

Slide 16

Preparation of standard solution of

sodium benzoate (10 ppm)

Preparation of standard solution of

Caffeine (10 ppm)

Weigh Sodium benzoate 100 mg

(Using analytical balance)

Weigh Caffeine .100 mg

(Using analytical balance)

100 ml

Dilute up to 100 ml
with water (1000 ppm)

100 ml

Use 1 ml pipette
100 ml

Take 1 ml from above

solution and dilute up
to 100 ml with water

Resulting solution
((10 pp
ppm))

Dilute up to 100 ml
with water (1000 ppm)
Use 1 ml pipette

100 ml

Take 1 ml from above

solution and dilute up
to 100 ml with water

Resulting solution
(10 ppm)

For injection
Anhydrous
y
caffeine..125 mg
g
Sodium benzoate ..125 mg
Water to
. 100 ml

Take 1 ml injection
l ti and
d dil
dilute
t up
solution
to 100 ml with water
Slide 17

Recording of overlain UV spectra of sodium benzoates and caffeine for

Simultaneous Equation method
Sodium benzoate (10 ppm)
max = 227 nm

Sod.Ben
nz.
Cafffeine.

Caffeine (10 ppm)

max = 273 nm

Blank

I
Injection

0.3

Injection (Diluted)

Sample cell

A1
Absorb
bance

Blank
Bllank

1.0

A2

ax1

ay1

ay2
ax2
Wavelength

Overlain spectra of Sodium benzoate and

C ff i
Caffeine
Slide 18

Absorbance ratio method

It is a modification of the simultaneous equation
q
p
procedure. It depends
p
on the
property that for a substance, which obeys Beers law at all wavelengths, the ratio
of absorbance at any two wavelengths is a constant value independent of
concentration or path length.
e.g. Two dilutions of the same substance give the same absorbance ratio A1 / A2.
In the USP, this ratio is referred to as Q value.
In 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 and the other being a wavelength of equal absorptivity of
the two components ii.e.,
e the iso-absorptive point.
point
1

Absorban
nce

Iso absorptive point

Wavelength

Slide 19

Two equations are constructed as described for the method of simultaneous

equation. Their treatment is somewhat different.
Consider

ax1 = ay1
At 1

at 1.

Assume b = 1 cm

A1 = ax1 bcx + ayy1 b cY since

(1)

A1 = ax1cx + ax1cY
A2
A1

ax1 = ay1

ax2 cx + ay2 cY
=

ax1cx + ax1cY

Divide each term by Cx + Cy and Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy)

Fx and Fy are the fractions of X and Y respectively in the mixture.
ax2 Fx + ay2 Fy
A2
=
A1
ax1 Fx + ax1 Fy

But Fyy = 1 - Fx

A2
A1

ax2 Fx + ay2 (1 Fx)

ax1 (Fx + Fy)

Fx ax2 - Fx ay2 + aY2

=

ax1
Slide 20

A2
A1

Fx ax2 - Fx ay2 + aY2

ax1
Let

Qx =

ax2
ax1

Fx ax2
ax1

Qy =

ay2
ay1

Fx ay2
ay1

QM =

aY2
ay1

A2
A1

QM = Fx (QX - QY) + QY
Fx =

(QM - QY)
(QX - QY)

Above equation gives fraction

fraction, rather than the concentration of X in the mixture in
terms of absorbance ratios. As these are independent of concentrations, only
approximate, dilutions of X and Y and the sample mixture are required to
determine QX, QY, and QM respectively.
For absolute concentration of X and Y, eq. (1) is rearranged
A1 = ax1 (Cx +CY)
Cx +CY

A1
ax1

Slide 21

Cx
Cx +CY
Cx
A1

=
ax1
Cx =

(QM - QY)
(QX - QY)
(QM - QY)
(QX - QY)
(QM - QY)

A1

(QX - QY)

ax1

Above equation gives the concentration of X in terms of absorbance ratios, the

absorbance of mixture and the absorptivities of the compounds at the isoabsorptive
wavelength Accurate dilutions of the sample solution and of the standard solutions
wavelength.
of X and Y are necessary for the accurate measurement of A1 and aX1 respectively.
Iso-absorptive point
It is defined as the point at which two different solutions of same concentration
shows same absorbance.

Slide 22

Assay of caffeine and sodium benzoate injection by the simultaneous

equation method
The composition of injection is

Anhydrous
A
h d
caffeine..125
ff i
125 mg
Sodium benzoate ..125 mg
Water to
. 1 ml

Dilute injection,100 times

Anhydrous caffeine..125 mg
Sodium benzoate ..125
125 mg
Water to
. 100 ml

Slide 23

Preparation of standard solution of

sodium benzoate (10 ppm)

Preparation of standard solution of

Caffeine (10 ppm)

Weigh Sodium benzoate 100 mg

(Using analytical balance)

Weigh Caffeine .100 mg

(Using analytical balance)

100 ml

Dilute up to 100 ml
with water (1000 ppm)

100 ml

Use 1 ml pipette
100 ml

Take 1 ml from above

solution and dilute up
to 100 ml with water

Resulting solution
((10 pp
ppm))

Dilute up to 100 ml
with water (1000 ppm)
Use 1 ml pipette

100 ml

Take 1 ml from above

solution and dilute up
to 100 ml with water

Resulting solution
(10 ppm)

For injection
Anhydrous
y
caffeine..125 mg
g
Sodium benzoate ..125 mg
Water to
. 100 ml

Take 1 ml injection
l ti and
d dil
dilute
t up
solution
to 100 ml with water
Slide 24

Absorb
bance

Bllank

max = 273 nm

Sod.Ben
nz.

Caffeine (10 ppm)

Cafffeine.

Blank

Recording of overlain UV spectra of sodium benzoates and caffeine for Ratio

Absorption Method
Sodium benzoate (10 ppm)
1
2
max = 227 nm
1.0

A2
ax1

A1
ay2
ay1

Blank

I
Injection

0.3

Injection (Diluted)

Iso-absorptive
point

ax2

Wavelength
Overlain spectra of Sodium benzoate and
C ff i
Caffeine

Sample cell
Slide 25

Derivative Spectrophotometry
Derivative spectrophotometry involves the conversions of a normal spectrum to its
first second or higher derivative spectrum
first,
spectrum.
In the context of derivative spectrometry, the normal absorption spectrum is referred
to as the fundamantal, zeroth order or D0 spectrum.
The first derivative (D1) spectrum is a plot of the rate of change of absorbance with
wavelength against wavelength.
At 2 and 4 , the maximum positive and maximum
4 5
1 2
0 spectrum correspond
negative
slope
respectively
in
the
D
3
A
with a maximum and a minimum respectively in the D1
spectrum.
The max at 3 is wavelength of zero slope and gives
dA
dA/d = 0, i.e. cross-over point, in the D1.
d
The second derivative (D2) spectrum is a plot of the
curvature of the d2A/d2 vs .

The maximum negative curvature at 3 in the D0 spectrum

gives a minimum in a D2 spectrum.
spectrum The maximum positive
+
curvature at 1 and 5 in the D0 spectrum gives two small
2 spectrum.
d2A
maxima
called
satellite
bands
in
the
D

d2
At 2 and 4 the wavelength of maximum slope and zero
curvature in the D0 spectrum correspond with cross-over
points in the D2 spectrum.
Slide 26

Advantages of Derivative Spectrophotometry

Spectral transformation confers two principal
spectrophotometry.
spectrophotometry

advantages

on derivative

1
An even order spectrum is of narrower spectral bandwidth than its fundamental
spectrum A derivative spectrum therefore shows better resolution of overlapping
spectrum.
bands than the fundamental spectrum and may permit the accurate determination
of the max of the individual bands.
Mixture of X and Y
X
Y

d2A
d2

(a)

(b)

Fig.:: (a) The individual spectra of two components X and Y in admixture and their
Fig
combined spectrum and (b) Second derivative spectrum of the mixture.
Slide 27

2
Derivative spectrometry discriminates in favour of substances of narrow spectral
bandwidth against broad bandwidth substances. This is because derivative
amplitude (D) i.e. distance from maximum to minimum, is inversely proportional to
the fundamental spectral bandwidth (W) raised to the power (n) of the derivative
order.
Derivative amplitude (D) = (1/Spectral width) n
The enhanced resolution and bandwidth discrimination, permit the selective
determination of certain absorbing substance in samples in which non specific
interference may prohibit the application of simple spectrophotometric methods.
Example: Ephedrine Hydrochloride
1%

Weakly absorbing substance (A1cmabout 15) at 240 270 nm

High excipent / drug ratio (typically 1 50 mg / unit dose)
High sample weight require for assay may introduce irrelevant absorption from the
formulation excipients
Second derivative spectrophotometry discriminates in favor of the narrow band of
the fine structure of the drug and eliminates the broad band absorption of the
excipients.

Slide 28

Limitation:
The enhanced resolution and bandwidth discrimination increases with increasing
derivative order. However, it is also found that there is increase in electronic noise
along with the increase in the derivative order, which place serious practical
limitations on the higher order spectra.
For quantative purposes, second and forth derivative spectra are the most
frequently employed derivative orders.

Slide 29

Example:
Brand name: Olmat H
Marketed by: Sun Pharma Ltd.
Olmisartam Medoxomil.20.00 mg
Hydrochlorothiazide. 12.50 mg

Overlain
O
l i UV-spectra
UV
t off OLM and
d HTZ (each
(
h 10 g/ml)
/ l) in
i
0.1 N NaOH solution.

Overlain
O
l i I Derivative
D i ti S
Spectra
t off OLM (10g/ml)
(10 / l) and
d HTZ
(10g/ml) in 0.1 N NaOH solution where n=4.

Overlain II Derivative Spectra of OLM (10 g/ml) and

HTZ (10g/ml) in 0.1 N NaOH solution where n=4.

Overlain III Derivative Spectra of OLM (10 g/ml) and

HTZ (10g/ml) in 0.1 N NaOH solution where n=4. Slide 30

Difference Spectrophotometry
Difference spectrophotometry improves selectivity and accuracy of the analysis of
compound of interest in presence of absorbing interferents.
It measures the value , which is difference of absorbance (A) between two
equimolar solutions of the analyte in different chemical forms which exhibit different
spectral
t l characteristics.
h
t i ti
Following criteria should be followed for difference spectrophotometry
a)) Reproducible
R
d ibl changes
h
can b
be produced
d
d iin th
the spectra
t off analyte
l t b
by th
the addation
dd ti
of one or more reagents.
b) The spectra and the absorbance of the interfering substance should not get
affected by the reagent.
reagent
The simple and most commonly employed technique for altering the spectral
properties of the analyte is the adjustment of the pH by means of aqueous solution
of acid,
acid alkali or buffers
buffers.
The ultraviolet-visible absorption spectra of many substance containing ionizable
functional groups, e.g. phenol, aromatic carboxylic acids and amines, are
dependent on the state of ionization of the functional groups and consequently on
the pH of solution.
Slide 31

Example: Phenylephrine hydrochloride

The ionization of the phenolic group in alkaline solution
generates an additional n (non-bonded) electron that interacts
with the ring electrons to produce bathochromic shift of the
max from 271 nm in acidic solution to 291 nm and increase in
absorbance at the max (hyperchromic effect)
Phenylephrine hydrochloride

Absorban
nce

0.8
278
257

0
250 270 290

310

Wavelength
g ((nm))

Absorption spectra of equimolar solutions of Phenylephrine hydrochloride in

a) 0.1 M NaOH (pH = 13) b) 0.1 M HCl (pH = 1)
Slide 32

The difference absorption spectrum is a plot of difference in absorbance between

the solution at pH=13 and that pH =1 against wavelength.
It may be
b generated
t d automatically
t
ti ll using
i double
d bl b
beam recording
di
spectrophotometer
t h t
t
with a solution at pH=13 in sample cell and the solution at pH=1 in reference cell
(blank) cell. At 257 and 278 nm both solutions have identical absorbance. Such
wavelength
g
of equal
q
absorptivity
p
y of the two analytes
y
are called isobestic or
isoabsorptive points.
Above 278 nm the alkaline solution absorbs more intensely than the acidic solution
and ((A)) is therefore positive. Between 257 to 258 nm it has negative
g
value.
2
0.8
A1
A2

0
-0.2

1
250 270 290

310

Difference absorption spectra of Phenylephrine hydrochloride of solution

0.1 M HCl (pH = 1) relative to 0.1 M NaOH (pH = 13)

Slide 33

The measured value in quantative difference spectrophotometric assay is the A at

any suitable wavelength measured to the baseline, e.g. A1 at 1 or amplitude
between adjacent maximum and minimum, e.g. A2 at 2
At

A1 = A alk A acid

Where Aalk and Aacid are the individual absorbances at 1 in 0.1 M sodium hydroxide
and 0.1M hydrochloric acid solution respectively.
If individual absorbances
absorbances, Aalk
lk and Aacid
id, are proportional to the concentration of
analyte and pathlength, the, A also obeys the Beer-Lambert Law and modified
equation may be derives as
A = abc
a is the difference absorptivity of the substance at the wavelength of measurement
If one or more other absorbing substances is present in the sample which at
analytical
l ti l wavelength
l
th h
has id
identical
ti l absorbance
b b
(Ax) in
i th
the alkaline
lk li
and
d acidic
idi
solutions, its interference in the spectrophotometric measurement is eliminated.
A = (Aalk + Ax) (Aacid + Ax)
= Aalk - Aacid

Slide 34

A substance whose spectrum is unaffected by changes of pH may be determined

by difference spectrophotometric procedure if it can be quantitatively converted by
means of suitable reagent to a chemical species that has different spectral
properties to its unreacted parent substance. The between eqimolar solutions of
the unreacted substance and its derivative is free of interference if the irrelevant
absorption is unaffected by the reagent.

Slide 35

Assay of Phenylephrine Hydrochloride in Drosyn eye drops

Brand Name: Drosyn 10% Eye Drops
Marketed by: FDC ltd.
Rx
Phenylephrine Hydrochloride IP
IP10%
10% w/v
Benzalkonium Chloride NF.0.01% w/v
A) Preparation of standard stock solutions of Phenylephrine Hydrochloride
Weigh Phenylephrine Hydrochloride..100 mg
(Using analytical balance)

100 ml

Dissolve & dilute it up

to 100 ml with water
(1000 ppm)

Label as Stock Solution for standard

Slide 36

100 ml

Stock Solution for standard

Take 1 ml from above

solution and dilute up
to 25 ml with water

Take 1 ml from above

solution and dilute up
to 25 ml with 0.1N HCl

Use 1 ml pipette

Use 1 ml pipette

Standard A

Standard B

25 ml

40 ppm

25 ml

40 ppm

Take 1 ml from above

solution and dilute up to
25 ml with 0.1N NaOH
Use 1 ml pipette

Standard C

25 ml

40 ppm
Slide 37

B) Preparation of sample solution of Phenylephrine Hydrochloride

y 10% Eye
y Drops
p
Brand Name: Drosyn
Marketed by: FDC ltd.
Rx
Phenylephrine Hydrochloride IP10% w/v
Benzalkonium Chloride NF
NF.0.01%
0 01% w/v
Use 1 ml bulb pipette

Take 1 ml from above

solution and dilute up
to 100 ml with water

Label as Stock Solution for sample

Take 1 ml from above

solution and dilute up
to 25 ml with 0.1N HCl
Use 1 ml bulb pipette

Take 1 ml from above

solution and dilute up to
25 ml with 0.1N NaOH
Use 1 ml bulb pipette

Sample D
40 ppm

Sample E
40 ppm

Slide 38

Recording of overlain UV spectra of standard A, standard B, standard C for

Difference spectrophotometric analysis

40 ppm

Standard A (water)
Standa
ard B (0.1N HC
Cl)
Standard
d C (0.1N NaOH
H)

Standard C

Blank (0.1N NaOH)

40 ppm

1.0

Absorb
bance

Blank (water)

40 ppm
Standard B

Blan
nk (0.1N HCl)

Standard A

Standard B
Isoabsorptive
points

Standard C

0.3
Wavelength (nm)
Overlain spectra of standard A, standard B
and
d standard
t d dC
Slide 39

Calculations

1.0

Absorb
bance

1 = 238.32
238 32 nm
Use A = abc
Standard B
0.0251 = aB b(1cm)
(
) 40 ppm
pp
aB = 0.00063
Standard C
1.8473 = aCb(1cm) 40ppm
aC = 0.0462
0 0462
a = aC aB = 0.04557

1 = 291.63 nm
Use A = abc
Standard B
0.01141 = aB b(1cm) 40 ppm 0.3
aB = 0.000285
0 000285
Standard C
0.57405 = aCb(1cm) 40ppm
aC = 0.01435
a = aC aB = 0.01406

Standard B

2
AstdC = 1.8473

AstdC = 0.57405
AstdB
0 0251
tdB = 0.0251

Standard C
AstdB = 0.01141

238.32
291.63
Isoabsorptive points
Wavelength (nm)
Overlain spectra of standard A, standard B
and standard C
Slide 40

Recording of overlain UV spectra of standard and sample for Difference

spectrophotometric analysis
Before recording difference spectrum, baseline correction was done by placing
0.1N HCl in the reference cell and 0.1N NaOH in the sample cell

Absorrbance

Standard C (0.1N NaOH

H)
Standard E (0.1N NaOH)

For sample:

Standard
d B (0.1N HCl)

For standard:

Standard
d D (0.1N HCl)

After the blank difference 1.8

18
spectrum was recorded for
standard as well as for sample

1
Peak 1, 238.32nm, A=1.834

Peak 2, 291.63nm, A=0.5656

2

0.3
Wavelength (nm)
Overlain spectra of standard and sample
Slide 41

Calculations

1.8343 = 0.04557b(1cm)C1
C1 = 40.21 ppm
% of Phenylephrine
= 40.21/40.0 *100 =100.5%
1 = 291.63 nm
Use A2 = a2bc2
0.5656
0
5656 = 0.0141(1cm)C
0 0141(1cm)C2
C2 = 40.12 ppm
% of Phenylephrine
= 40.12/40.0 *100 =100.3%

18
1.8

Absorrbance

1 = 238.32 nm
Use A1 = a1bc1

1
Peak 1, 238.32nm, A=1.834

Peak 2, 291.63nm, A=0.5656

2

0.3
Wavelength (nm)
Overlain Difference spectra of standard and
sample
Slide 42

Chemical Derivatization
Indirect spectrophotometric assay are based on the conversion of the analyte by a
chemical reagent to a derivative that has different spectral properties.
When an excess of the reagent is used, to ensure complete conversion, the
absorbance of derivative is usually, but not always, proportional to the conversion of
the analyte. The majority of the procedures involve the conversion of the analyte to
a derivative that has a longer max and / or higher absorptivity.
Chemical derivatization may be adopted for any of several reasons
a) if analyte absorbs weakly in the ultraviolet region, a more sensitive method of
assay is obtained by converting the substance to a derivative with more intensely
absorbing chromophore.
A h
Anthrone
Colour derivative of Sugars
Sugars
which absorb more intensely at
Do not absorb more intensely above Sulphuric acid
625 nm
220 nm

b) The interference from irrelevant absorption may be avoided by converting the

analyte to a derivative which absorbs in the visible region, where irrelevant
absorption is negligible.
e.g. The condensation of ketosteroids with hydrazide reagent or oxidation of alphaalpha
ketol group by tetrazolium salts, produce derivatives that absorb in the visible
region, free of interference from irrelevant absorption

Slide 43

C) Indirect spectrophotometric procedure are also used to improve the selectivity of

the assay of an ultraviolet absorbing substance in a sample that contains other
ultraviolet
lt i l t absorbing
b bi components.
t
e.g. The assay of low concentration of adrenaline in Procaine and Adrenaline
Injection by a direct measurement of absorbance at the absorption maximum of
adrenaline around 279 nm is subject
j
to g
gross interference from the bactericidal
chlorocresol and from high concentration of procaine hydrochloride. Only adrenaline
forms the purple derivative in the presence of iron (II) and is measured
colorimetrically free of interference from the other UV absorbing components
d) The adoption of visible spectrophotometric procedure, instead of an ultraviolet
procedure, may be based on cost considerations.
e.g. Colorimeters are much cheaper that uv-visible spectrophotometer.

Slide 44

Diazotisation and coupling of primary aromatic amines

The amine is first diazotised with an aqueous solution of nitrous acid (generated in
situ by the reaction of hydrochloric acid and sodium nitrite) at 0 to 50c
ARNH2+HNO2

ARN+N+2H2O

H+

The colorless diazonium salt is very reactive and when treated with a suitable
coupling reagent (Ar H) , e.g. a phenol or aromatic amine, undergoes an
electrophillic substitution reaction to produce an azo derivative.
A N N
Ar-N+
N+A
Ar-H
H

A
Ar-NN
N N Ar
A +H
H+

The azo derivative are coloured and consequently have an absorption maxima in
visible region. The max and max depends on the Ar and Ar groups. Among
the most widely used coupling reagents are 1
1- naphthol , 2
2-naphthol
naphthol and N-(1N (1
napthyl)-ethane 1,2-diammonium dichloride( the bratton- marshall reagent) which
gives high absorptivities. For example, the azo derivative of diazotized
sulphadiazine coupled with the bratton marshall reagent is
N
NHSO2

NHCH2CH2NH2

Which absorbs intensely around 545 nm owing to its excessive conjugation.

Slide 45

The sensitivity and selectivity of the procedure permit the assay of low
concentration of impurities that contain a primary amine group in the presence of
th parentt substance
the
b t
lacking
l ki amine
i ffunction.
ti
B
British
iti h pharmacopoeia
h
i ttests
t ffor ffree
amine impurities in frusemide, Iothalamic acid and Iodipamide meglumine injection
are based upon diazotization and coupling. Substances can be hydrolysed
((e.g.chloramphenicol)
g
p
) to derivative containing
gap
primary
y aromatic amine g
group
p can
also be assayed spectrophotometrically by diazotization and coupling of the amine
derivative.
Condensation reactions
Many colorimetric procedure are based on the rapid reaction that occurs under
suitable condtion between amines and carbonyl compounds. The reaction involve
nucleophilic attack by the amine on carbonyl carbon with the elimination of water.
S b t
Substances
containing
t i i a carbonyl
b
l group reactt with
ith a variety
i t off reagents
t containing
t i i
a amino group:
R'

R'

C
R''

NH2R'''
R''
R

NR'''

H2 O

When R =alkyl or aryl, the products is a schiffs base

=NH2 (HYDRAZINE), The product is a hydrazone
=NHCONH
NHCONH2 (semicarbazide),
(
i b id ) th
the product
d t iis a semicarbazone
i b
=OH(hydroxylamine), the product is an oxime
Slide 46

A number of hydrazine and hydrazide reagents have been described for the
colorimetric assay of ketosteroids. The selectivity of the reactions for steroids with
the keto group in different position depends on the reagent
reagent. Isoniazid (isonicotinic
acid hydrazide) reacts with 4-en-3-one and 1,4-dien-3-one steroids in acidic solution
to form yellow derivatives with max around 400nm.
A
CONHNH2 +

CONHN

Amino compounds can be assayed spectrophotometrically using a suitable

carbonyl reagent . One of the most frequently used reagents is 4dimethylaminobenzaldehyde(Ehrlichs reagent) used, for example, In the assay of
procaine
i h
hydrochloride
d hl id iin L
Lymecycline
li
and
d procaine
i
iinjection.
j ti
Th
The condensation
d
ti
product which absorbs at 454 nm is

(CH3)N

CH=N

COOCH2CH2N(C2H5)2

Slide 47

Reduction of tetrazolium salts

In the presence of steroids with an -ketol (21-hydroxy-20-keto) side chain group,
tetrazolium salts are reduced to their coloured formzan derivatives. Several
formulation containing corticosteroids are assayed using triphenyltetrzolium
chloride. The reaction is carried out in an alkaline medium (Tetramethylammonium
hydroxide) at 3030 350 for 1-2
1 2 hrs. and the absorbance of the red product is
measured around 485 nm. The oxidation of the -Ketol and the reduction of
triphenyltetrazolium chloride to triphenylformazan are shown below

CH2OH
C

O
OH

C6H5

C6H5

C6H5

Cl

HC
C

O
O

OH
D

C6H5

NH
C6H5

+ HCl

C6H5

Slide 48

The acid-dye method

The addition of an amine in its ionised form to an ionised acidic dye,e.g. methyl
orange or bromocresol purple,yields a salt (ion-pair) that may be extracted into an
organic solvent such as chloroform or dichloromethane.The indicator dye is added
in excess and the pH of the aqueous is adjusted if necessary to a value where both
the amine and dye are in ionised form.The ion-pair
ion pair is seperated from the excess
indicator by extraction into the organic solvent and the absorbance is measured at
the max of the indicator in the solvent.
The acid-dye method therefore provides a more sensitive technique for certain
amines and quaternary ammonium that absorbs weakly in ultraviolet region, e.g.
hyoscine butylbromide (a=202).
Oxidation methods
Oxidation of the side chain of weakly absorbing compounds containing a simple
phenyl group produces a carbonyl derivative that has a much greater absorptivity
than the parent compound. Commonly used oxidation reagents are alkaline
potassium permanganate solution, acidified potassium dichromate solution, or
perchlorate solution.The product formed from simple monophenyl compounds e.g.
ephedrine or propanolamine
propanolamine, is the corresponding benzaldehyde derivative
derivative, which
exhibits intense absorption, at around 240 nm owing to the interaction of the
carbonyl pie electrons with the ring electrons.

Slide 49

The assay of Ephedrine Hydrochloride Elixir involves the extraction of the

benzaldehyde into cyclohexane, and measurement of the absorbance at its max
241nm.
241nm
OH
NHCH3

CH3

Ephedrine

Benzaldehyde

Metal Ligand Complexation

Many organic reagents (in this context called ligands) form complexes with metal
atoms by the formation of coordinate bonds (in which both electrons are donated by
the ligand) and covalent bonds. Ligands with two or more donating groups may
share
h
more th
than one pair
i off electrons
l t
with
ith a single
i l metal
t l atom
t
by
b coordinating
di ti
tto
two or more positions. The chelates formed by these multidentate ligands with metal
atoms are often coloured, and consequently their concentration may be determined
byy visible spectroscopy.
p
py
Characteristic colours which vary in hue with change of pH are given, by the
reaction of iron(2)ions with phenols that contain two adjacent hydroxyl groups. Thus
g a solution of adrenaline with a buffered solution containing
g iron ((II))
on mixing
sulphate, a purple complex (max 540nm)is formed that has a maximum intensity at
pH 8-8.5.
Slide 50

OH
O
Fe
O

C
H

CH2NHCH3

++

S 4 -So

Slide 51

Thank You for your attention!