INDIRECT

SPECTROPHOTOMETRIC
METHODS

TO BE PRESENTED BY:
N.T.ARUN,
FIRST M.PHARM,
DEPARTMENT OF PHARMACEUTICAL ANALYSIS.

EVALUATORS:
1. Mrs. Suganthi .A - M.Pharm (Ph.D.,) Lecturer-Dept. of Pharma. Analysis,
2. Mrs. Manjuladevi.A.S.- M.Pharm (Ph.D.,) Asst.Professor-Dept.of Pharmacy Practice,
3. Mrs. M.Dhanamani - M.Pharm (Ph.D.,) Lecturer-Dept. of Pharmacognosy.
 Spectrophotometric methods

The spectrophotometric methods to be discussed (methods of molecular

absorption spectrometry) are based on the measurement of absorption of
radiation, in the visible and near ultraviolet regions, owing to coloured
compounds formed, before the determination, by the elements to be
determined. Only seldom is use made of the intrinsic colour of the
element i t se l f , in its ionic form. In cases where an element neither
forms coloured compounds nor occurs in a coloured form, indirect
spectrophotometric methods are applied.

Spectrophotometric methods are characterized by high versatility,

sensitivity, and precision. They may be used for the determination of almost
all chemical elements over a wide range of concentrations, from macro
quantities (by means of differential spectrophotometry) to traces ranging
from 10-6-10-8% (after suitable preconcentration).Spectrophotometric
methods are among the most precise instrumental methods of chemical
analysis. The advantages mentioned of the spectrophotometric methods are
made greater by their availability. A spectrophotometer, which is the basic
instrument in this field, is cheaper than most other fundamental instruments
used in chemical analysis.

Indirect spectrophotometry
This is also known as chemical derivatisation method or colorimetric
method. Indirect spectrophotometric assays are based on the conversion of
the analyte by a chemical reagent to a derivative that has different spectral
properties, thereby shifting λmax towards longer λ.

Chemical derivatisation is adopted because

1. If the analyte absorbs weakly in the UV region, a more sensitive method

of assay is obtained by converting the substance to a derivative with a
more intensely absorbing chromophore.
Ex: sugars don’t absorb above 220nm. This can be determined
spectrophotometrically by heating with anthrone in
conc.sulphuric acid and measuring absorbance of
 coloured derivatives at 625nm.
2. 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.
Ex: The condensation of ketosteroids (methyl testosterone in methyl
testosterone tablets with hydrazide reagents produces derivative that
absorb in the visible region free of interference from irrelevant
absorption.
3. It is used to improve the selectivity of the assay of an UV absorbing
substance in a sample.
Ex: In the assay of adrenaline in adrenaline injection by direct
measurement, the maximum absorption is obtained at 279nm. It’s
subject to gross interference due to bactericidal chlorocresol. Only
adrenalin forms purple derivative in the presence of iron (II) is
measured colorimetrically.
4. The adsorption of a indirect spectrophotometric procedure, instead of
an UV procedure, may be based on cost consideration. Single beam
manually adjusted visible spectrophotometers are much cheaper than
UV spectrophotometer.
5. Apart from these, we have to evaluate some drugs by this method like
Dicyclomine.Hcl, pempidine, piperidine ring structure drugs, ephedrine,
amphetamine , atropine, metals like iron(using 1,10-phenanthroline),
lead (using dithiazone), etc.

In the usual direct spectrophotometric methods of analysis, the absorbance

of substance or derivative being determined is measured. The color-
producing reaction may be illustrated:
Sample (a1) + chromogenic reagent (a2) -product (a3)
Where a represents the absorptivity of the species in question. Generally, a1
and a, are zero at the wave length employed in the analysis, and a, is
fairly large in order that the method be sensitive. In the usual
spectrophotometric method, a3 >> a1 or a2.Because the absorbance is
directly proportional to the concentration of the colored sample or its
coloured derivative, the analysis is simple and straightforward. In
indirect spectrophotometry, the sample is first allowed to react with a
definite concentration of a highly colored chromogenic reagent, present in
excess. The product of this reaction is of less intense color than the original
chromogenic reagent (a3< a2) and consequently, a fading of the chromogenic
reagent solution occurs. The extent of fading is a direct measure of the
quantity of sample present. Several modes of photometric measurement for
the extent of fading are applicable, each method having its own advantages
and disadvantages.

CLASSIFICATION OF INDIRECT SPECTROPHOTOMETRIC METHODS

Photoelectric measurements have in common the preliminary adjustment of

the instrument to read first O%T and then 100%T, under specified
conditions. The photometric methods described here differ essentially in the
types of reference solutions used to establish the 100%1 setting. Figure 1
summarizes four methods described below.

Method A;
The instrument is set to read O%T with the photocell in darkness and
100%T when exposed to light which has passed through the pure solvent.
After these preliminary adjustments, the faded sample-chromogenic product
is read, this reading falling between O%T and 100%T.
The resulting plot of absorbance vs. concentration is actually a photometric
titration, and a typical curve is shown in Figure 2. In this figure the solid
lines represent the situation obtained when the sample and chromogenic
reagent react completely.
In practice, however, the dotted line is more often obtained because of
incomplete reaction between the sample and the chromogenic reagent.
 At low concentrations of sample the absorbance reading is rather
high, with a concomitant larger error in reading the absorbance scale, as it
is crowded at high values. Consequently, better results are obtained at
the low absorbance readings corresponding to higher sample concentration
values. When the sample and chromogenic reagent react almost completely-
i.e., a case of favor­ able equilibrium-the lowest relative error occurs at low
absorbance readings. In fact, the point of minimum error actually occurs at
the intercept.
When the concentration of sample becomes sufficiently great and
rounding of the curve commences because of equilibrium considerations,
error in the analysis will increase in this region because the change in
absorbance for a given change in concentration decreases considerably.
The point of minimum error will occur at an intermediate concentration
removed from the two areas of high error, the region near zero con­
centration and the region where the absorbance per unit change in
concentration becomes small.

Method B;
This case is a modification of Method A. The instrument is set to read O%T
with the photocell in darkness and 100%T when exposed to light which has
passed through the solution of chromogenic reagent that has been partially
bleached by the introduction of a standard quantity of the sample material.
After these two initial settings the unknown sample is read, the value of
the reading falling between O%T and 100%T. The amount of standard
sample introduced into the reference solution used to set 100%T on the
instrument must be somewhat higher than the sample to be read.
A typical calibration curve for this mode of analysis is shown in Figure 3.
Although no readings can be actually obtained below zero, these off-scale
readings are included to illustrate that the response now obtained is simply
an expansion of the scale illustrated in Figure 2. This effective expansion of
the absorbance scale diminishes the error in the analysis.

Method C;
This method, presumably heretofore unmentioned, employs a very unusual
method for setting the instrument. The instrument is set to read O%T with
the photocell in darkness and IOO%T when exposed to light which has
passed through the sample-chromogenic reagent solution. After these
settings, the pure chromo­ genic reagent absorbance is read, the reading
falling between O%T and100%T. Operation in this way will yield data
of the type represented in Figure 4.
If the reaction between the chromogenic reagent and the sample species is
complete, then a sharp break at the equivalence point, E.P., will be
obtained.
Where equilibrium is not so favorable, rounding Occurs, illustrated by
the dotted line in Figure 4.In practice, only the straight-line portion prior to
E.P. is employed. The range of the straight line can be extended simply by
increasing the concentration of chromogenic reagent employed, causing
the E.P. to occur at higher concentrations of sample
This particular method of operation is particularly useful for low
concentrations of sample, because the absorbance readings are obtained in
that section of the absorbance scale where the scale readings are well
separated and read with higher accuracy.
However, each sample requires a new slit width setting, making
Method C somewhat more in­convenient and time-consuming than Method
A. In addition, it may cause deviations from Beer's law, but this will not
affect the analytical results because a calibration curve is usually employed.

The absorbance of the pure chromogenic reagent solution referred to water

(or to pure solvent) is given by:

A1=a1bC1………………………………… (1)

Similarly the absorbance of the unknown sample referred to water is given

by:

A1=a1b (C1-nC2) +a2bn’C2………………… (2)

where C2 denotes the unknown concentration of the sample species, n the

moles of chromogenic reagent that react with each mole of sample species,
and n’ the moles of product formed from each mole of sample species. When
measuring the absorbance of the pure chromogenic reagent solution against
the sample, the absorbance is given by:

A = A1 - A2 = (aln – a2n‘) bC2 =a‘bC2 …………… (3)

A formula analogous to Beer’s law i.e., absorbance directly proportional to

concentration-is obtained. The a’ value is determined by the difference
between the absorptivity of the chromogenic reagent and that of the sample
chromogenic reagent product, each multiplied by the number of moles of
chromogenic reagent or product consumed or formed per mole of sample
species.
Because of the formal similarity between the Beer’s law equation
and Equation 3, the usual treatment of photometric errors can be applied (3,
5,9,12,13). For example, if a straight line is obtained as predicted by
Equation 3, one would expect lowest relative error at sample concentrations
where the absorbance reading is 0.43. If the calibration line is curved, one
should plot 1-T vs. log C (Ringbom plot) in order to calculate the
photometric error expected at any particular concentration value and to
determine the useful concentration range (1,3, 11)

Method D;
This method is essentially a modification of Method C and resembles the
transmittance-ratio method. The instrument is set to read O%T with the
photocell in darkness and l00%T when exposed to light which has passed
through a solution containing the chromogenic reagent and a certain amount
of unknown sample. The absorbance of a solution containing the reagent and
a fixed amount of standard sample is then read. The concentration of
standard sample must be somewhat less than the concentration of the
sample to be determined. The calibration curves are illustrated as lines C
and D (Figure 4) where the reference solution contains known
concentrations, C3 and C3‘, respectively, of the sample species.
The equation for this method may also be derived. The
absorbance of the fixed amount of standard, C3, referred to water is given by:

A3 = a1b (C1 – nC3) + a2bn‘C3……………………… (4)

Combining this equation with Equation 2, one obtains the absorbance of the
fixed amount of reference standard referred to the unknown sample:

A = A3 – A2 = (a1n – a2n’) b (C2 – C3)……………… (5)

EXPERIMENTAL

Determination of Magnesium.
In the determination of magnesium, Method C is used. The chromogenic
reagent is Calcon [1-(2-hydroxy-lnaphthylazo) 2 -naphthol - 4 – sulfonic
acid; CI 202], which forms a stable chelate with magnesium (4). This chelate
has a different absorbance spectrum from that of the unchelated Calcon
(Figure 5) and at 620 mµ, where the free Calcon absorbs strongly, the
addition of magnesium decreases the concentration of free Calcon causing a
bleaching effect. From this bleaching effect a calibration curve can be
constructed using Method C. and a colorimetric method for magnesium is
thereby obtained. The color of the chromogenic reagent depends upon pH as
shown by the following equilibria:

pk2=4 pk3=13.5
-2
H2In- HIn In-3 + H+
Pink Blue Pink
The effective stability of the magnesium-Calcon chelate is also pH dependent
e.g., at pH 10 the reaction is essentially:

Mg+2 + HIn-2 MgIn- + H+

Blue Pink
Consequently buffer must be added to maintain a constant pH or
reproducible results are not obtained. The ammonium chloride-ammonium
hydroxide buffer system at pH 10 is satisfactory. At pH values below 10 the
stability of the magnesium-Calcon chelate decreases appreciably. At pH
values above 10 the colour stability of the chelate decreases, attributed to
hydrolytic attack and air oxidation of the Calcon. The quantity of buffer
added also affects the calibration curve. The amount used is a compromise
between the sensitivity- of the method and the buffer capacity of the
solution. The results are given in Table I.

Determination of Calcium:
Calcium cannot be determined in the same way as magnesium because the
Stability of the calcium-Calcon chelate is much less than that of the
magnesium-Calcon chelate. At pH 10 the reaction

Ca+2 + HIn- CaIn- + H+

does not proceed sufficiently far to the right to create the bleaching effect
required for a successful colorimetric determination.
This difficulty can be readily surmounted by adding an excess of
magnesium-EDTA to the buffer solution. The calcium then replaces the
magnesium from the magnesium-EDTA buffer solution:
Ca+2 + MgEDTA-2 Mg+2 + CaEDTA-2
The replaced magnesium ions then chelate with Calcon, giving rise to the
same bleaching effect that occurs in the magnesium determination. A similar
principle has been proposed by Menon and Das, but using Erio-T as
chromogenic reagent and Method A for photometric measurement.
The results in Table are obtained using Method C for the spectrophotometric
settings. These results were obtained after the solutions had cooled to room
temperature, approximately 1 hour, as the heat of reaction and heat of
dilution of the reagents cause a rise in temperature. Longer standing time
should be avoided, because after 3 hours the absorbance decreases.

Reagents and solutions:

Standard calcium solution, EDTA solution, Magnesium stock solution,

Calcium stock solution, Buffer solution A (NH4-NH4cl), Magnesium-EDTA
solution, Buffer solution B (NH4cl- NH4OH), Calcon.

Procedure for Magnesium:

Prepare a calibration curve by delivering 5-, l0-, 15-, and 20-ml.
aliquots of stock magnesium solution containing 2.4 γ of magnesium per ml.
into 50-ml. volumetric flasks. Into each flask pipette 5 ml. of buffer solution
A, 10 ml. of color reagent and dilute contents to volume with distilled water.
Also prepare a blank, omitting the magnesium. Make the sample solutions in
a similar manner, replacing the stock magnesium solution with the sample
aliquot. In cases where the sample contains excess acid or base,
preneutralize the sample before adding the buffer solution and color
reagent. Allow the solutions to stand to cool for approximately 1 hour and
again dilute to volume.
Set the spectrophotometer at 620 mµ. Place the blank solution and 5-
ml. stock magnesium solution in I-cm. cells. Set the dark current in the usual
manner with the shutter closed. Introduce the 5ml. stock magnesium
solution into the light path and set the instrument to read 100%, T by
adjusting the slit width. Put the blank solution in the light path and record
the absorbance. Repeat using lo-, 15, and 20-ml. stock solutions.
From the absorbance of the stock magnesium solutions. Construct a
calibration curve. The concentration of the sample is taken directly from the
curve.

Procedure for Calcium:

Prepare a calibration curve by delivering 5-, l0-, 15-, and 20-ml.
aliquots of stork calcium solution into separate 50-ml. volumetric flasks. To
each flask pipette 5 ml. of buffer solution B and 10 ml. of color reagent, and
dilute to volume with distilled water. Also prepare a blank solution, omitting
the calcium.
Prepare sample solutions in a similar manner, replacing the, stock calcium
solution with a sample aliquot. In cases where the sample contains excess
acid or base, neutralize the sample before adding the buffer solution and
color reagent. Allow the solutions to stand for 1 hour and again dilute to
volume. Read the stock calcium and sample solutions using the procedure
described for magnesium. From the recorded data prepare a calibration
curve and determine the concentration of the sample directly from the
calibration curve. A Beckman Model B spectrophotometer was used.

CHEMICAL DERIVATISATION:
In the early days of quantitative pharmaceutical analysis, chemical
reactions were involved in almost all the methods; titrimetry,
gravimetry, and colorimetry. Later, however, the role of chemical
reactions decreased considerably. The reason for this was the spread of
UV spectrometry and other spectroscopic techniques, as well as various
chromatographic methods. Using these techniques, chemical reactions can
be completely omitted (e.g. in spectroscopic methods, HPLC with UV or RI
monitoring, TLC densitometry based on natural absorption or
fluorescence), or the reaction takes place in the measuring cell or detector
only (e.g. in voltammetry methods, HPLC with electrochemical detectors,
gas chromatography with a flame ionization detector).

The role of derivatisation reactions in stationary or flowing systems is again

increasing. The reasons for this seem to be the following:
(a) Using preliminary chemical reactions the field of application of
spectroscopic and chromatographic methods can be greatly expanded:
spectrometrically inactive materials can be determined by UV-visible
spectrophotometry and by HPLC with UV detection
(b) The application of derivatisation reactions is sometimes a useful tool in
the spectroscopic or chromatographic identification of unknown
components.
(c) The main reason for the increasing use of derivatisation reactions is
certainly the continually increasing demand on pharmaceutical analysts for
more selective and/or sensitive methods
Even an outline summary of the vast number of papers in this field is beyond
the scope of this review: only the most important monographs dealing with
derivatisation reactions in chromatography and spectroscopy are listed.

 DIFFERENT METHODS:
 Diazotisation & Coupling.
 Condensation Reaction.
 Reduction of tetrazolium salts.
 Acid-dye method.
 Oxidation method.
 Metal-ligand complexation.

Derivatization of Carboxyl Groups for the Spectrometric Assays

The majority of carboxylic acids (among them drugs and biologically

important derivatives) are spectrometrically inactive or absorb only in the
UV region. Several derivatization methods are available for their UV-visible
spectrometric determination or for HPLC analysis with spectrophotometric
or fluorimetric detection. The aim of this study has been to develop a general
method for the transformation of carboxyl groups to carboxamide
derivatives with chromophoric or fluorophoric properties suitable for
spectrometric or HPLC determination.
The key reaction of the method is between the carboxylic acid and diethyl
chlorophosphite leading to a mixed anhydride

R-COOH + P (OC2H5)2Cl = R-CO-O-P (OC2H5)2 + HCl. (1)

This reaction takes place very rapidly at room temperature using

acetonitrile or a mixture of acetonitrile and tetrahydrofuran as solvent.
Diethyl chlorophosphite should be used in large excess to avoid interference
from water in the solvent (which should be under 0.05%) and to ensure
complete reaction. After completion of the reaction, any reactive and
spectrophotometrically active primary or secondary amine or hydrazine
can be added to form the corresponding carboxamide or hydrazide
derivative:

R-C0-0-P (OC2H5)2 + R'-NH2 = R-CO-NH-R' + P (OH)(OC2H5)2 (2)

Reaction (2) is somewhat slower than reaction (1), but it can be

completed at moderately elevated temperatures within 1-2 h, even with the
least reactive derivatives. At first aniline was used as the amine component
in a slow, single-step process, the carboxanilide derivatives having a strong
absorption band at 243 nm (e-14,500). Here the use of 2-
nitrophenylhydrazine as the amine component is described. This reagent
was for the detection of carboxylic acids and anhydrides and for the
quantitative determination of carboxylic anhydrides and chlorides. The use
of 2-nitrophenylhydrazine as a colorimetric reagent for the determination of
some water-soluble carboxylic acids was described. The carboxylic acids
were coupled witi1 the reagent with the aid of water-soluble
carbodiimides. The advantage of 2-nitrophenylhydrazine over aniline is that
in alkaline medium (pH>12) the acyl derivatives are highly conjugated with
absorption maxima around 550 nm, greatly increasing the selectivity of the
method.
 NH NH2 NH NH3+Cl-

NO2 NO2

P (OC2H5)2Cl 2

P(OC2H5)2 NH NH P (OC2H5)2

O O
NO2
C

O OH-

NH NH C R

NO2
OH

O- PO(OC2H5)2-

N N C R

N-O2

As is seen in reaction (3) the excess of diethyl chlorophosphite reacts with

the 2- nitrophenylhydrazine reagent and the product decomposes upon
addition of sodium hydroxide to form the colorless 1-hydroxy-1,2,3-
benztriazol. Both reactions are almost instantaneous and as a result of this
the reagent blank is negligibly small at 550 nm. By contrast isobutyl
chloroformate, which is more often used to form mixed anhydrides, cannot
be applied in this work because its reaction product with 2-
nitrophenylhydrazine does not decompose in alkali: this would cause
extremely high reagent blank absorption.
Several carboxylic acids have been determined by the proposed
method. The general procedure is the following.
The sample containing up to 5µl of carboxylic acid, is dissolved in 250 µl of
anhydrous acetonitrile or (in the case of bile acids) in a 7:3 v/v mixture of
acetonitrile and tetrahydrofuran. A 50µl portion of diethyl chlorophosphite
is added and the mixture is allowed to stand at room temperature in a well-
closed vial for 15 min. A 250 µl portion of 2-nitrophenylhydrazine reagent is
 then added (0.04 Min acetonitrile) and the mixture is heated at 700c for 30
min. The contents of the vial are then transferred to a 25 ml calibrated flask
with the aid of about 15 ml of ethanol. A 5 ml portion of 0.5 M aqueous
sodium hydroxide is added and the flask is made up to volume with ethanol.
The absorbance is read against a reagent blank at the absorption maximum
(ca. 550 nm). The absorbance is stable for several hours.

Table 1

Spectral data of carboxylic acids after derivatization with 2-nitrophenylhydrazine

Acid Amax (nm) Molar absorptivity• Regression datat a b

Acetic acid 550 6520±1.4% 0.008 1.072

Palmitic acid 550 6850±0.8% 0.004 0.270
Cholicacid 550 7280±1.3% -0.001 0.187
Benzoic acid 560 8030±1.2% 0.006 0.795

• Mean of eight determinations ± r.s.d.

T Regression equation: absorbance = a + b x concentration (mg/100 ml):r >0.9995.

Diazotisation & Coupling:

The amine is first diazotized with an aqueous solution of nitrous acid
(generated in situ by the reaction of hydrochloric: add and sodium
nitrite) at 0-50C
H+
Ar-NH2 + HN02 Ar-N+≡N + 2H2O
The colourless diazonium salt is very reactive and when treated with a
suitable coupling reagent (Ar'-H), e.g. a phenol or aromatic amine, undergoes
an electrophilic substitution reaction to produce an azo derivative.

Ar-N+≡N + Ar’-H Ar-N=N-Ar’ + H+

The azo derivatives are coloured and consequently have an absorption
maximum in the visible region. The λmax and € max depend on the Ar and Ar’
groups. Among the most widely used coupling reagents are 1-naphthol, 2-
naphthol and N-(1-naphthyl)-ethane-1, 2-diammonium.dichloride (the
Bratton -Marshall Reagent) which give high absorptivities. For example, the
azo derivative of diazotized sulphadiazine coupled with the Bratton-Marshall
reagent is

which absorbs intensely around 545 nm owing to its extensive conjugation.

Sulphamic acid or ammonium sulphamate is added to the solution of the
diazotised amine before the coupling stage to destroy the excess nitrous acid,
which inhibits the coupling reaction.

HN02 + NH2S03H N2+ H2S04 + H2O

The sensitivity and selectivity of the procedure permit the assay of low
concentrations of impurities that contain a primary aromatic amine group
in the presence of the parent substance lacking the amine function.
British Pharmacopoeia tests for free amine impurities in Frusemide, Iothalmic
acid and Iodpamide Meglumine Injection are based upon diazotisation and
coupling. Substances that can be hydrolysed (e.g. Bendrofluazide;
Chlorothiazide) or reduced (e.g. Chloramphenicol) to a derivative containing a
primary aromatic amine group can also be assayed spectrophotometrically by
diazotisation and coupling of the amine derivative.

Condensation Reaction:
Many colorimetric procedures are based on the rapid reaction that occurs
under suitable conditions between amines and carbonyl compounds. The
reactions involve the nucleophilic attack by the amine on the carbonyl carbon
with the elimination of water. Substances containing a carbonyl group react
with a variety of reagents containing an amino group:
 R' R'
NH2R''' NR''' H2O
C O C
R'' R''

When R"' = Alkyl or aryl, the product is a Schiff's base

= NH2 (hydrazine), the product is a hydrazone
= NHCONH2 (semicarbazide), the product is a semicarbazone
= OH (hydroxylamine), the product is an oxime
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 positions depends on the reagent.
Isoniazid (isonicotinic acid hydrazide) reacts with 4-en-3-oue and 1, 4-
dien-3-one steroids in acidic solution to form yellow derivatives with λ max
around 400 nm.

N CONHN
N CONHNH3 A
O

The reagent is used in the assays of Nandralone D e c a n o a t e Injection

and Betamethasone Sodium Phosphate Injection. A similar reagent 2,
4-dinitrophenylhydrazine, is used i n the a s s a y of M e t h y l testosterone
Tablets. The hydrazone formed on condensation of the 3-, 17- or 20-
Ketosteroid with (carboxymethyl) trimethylammonium chloride hydrazide
(Girard's reagent T) is water-soluble owing to the quaternary ammonium
group, and lipid-soluble impurities may b e removed by extraction
into chloroform. Alternatively, the hydrazone itself may be extracted i n t o
dichloroethane as the ion-pair with bromothymol blue.

Amino compounds can be assayed s p e c t r o p h o t o m e t r i c a l l y usi ng a

suitable carbonyl reagent. One of the most frequently employed re­
agents is 4-dimethylaminobenzaldehyde (Ehrlich's reagent) used, for
example, in the assay of procaine hydrochloride in Lymecycline and
Procaine Injection. The condensation product which absorbs at 454 nm is
H3C
N CH N COOCH2CH2N(C2H5)2
H3C

In the presence of a steroid with an α-ketol (21-hydroxy-20-keto) side-

chain group, tetrazolium salts are reduced to their coloured formazan
derivatives. Several formulations containing corticosteroids are assayed
using triphenyltetrazolium chloride. The reaction is carried out in an
alkaline medium (tetramethylammonium hydroxide) at 30-35° for 1-2 h
and the absorbance of the red product is measured around 485 nm. The
oxidation of the α-ketol group and the reduction of triphenyltetrazolium
chloride to triphenylformazan are shown:

Steroids esterified in the 21-position, e.g. Hydrocortisone Acetate, hydrolyse

in the alkaline solution to yield the free 21-hydroxysteroids and are also
determined by this procedure. Precautions are taken throughout the assay
against the effects of light and oxygen.
Acid-dye method:
The addition of an amine in its ionised form to an ionised acidic dye,
i.e.-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 solution is adjusted if necessary to a value where both the
amine and dye are in the ionised forms. The ion-pair is separated from
the excess indicator by extraction into the organic solvent, and the
absorbance is measured at the λmax of the indicator in the solvent.
Usually, the most intensely absorbing form of the indicator is measured,
with the addition, if required, of acidified or basified ethanol.
Alternatively, the absorbance of the indicator may be measured in
aqueous solution after back extraction from the organic solvent.
The molar absorptivities of the ion-pairs formed between
quaternary ammonium compounds and methyl orange or bromothymol
blue are typically 2 x 10 4 - 4 X 104. The acid-dye method therefore
provides a more sensitive technique for certain amines and quaternary
ammonium compounds that absorb weakly in the ultraviolet region,
e.g. Hyoscine butyl bromide (€ 257 = 202).
The correct choice of pH may permit the selective assay of
a mixture of an amine and a quaternary ammonium salt. For example, in
the assay of a tertiary base and a quaternary ammonium salt both
substances are ionic at pH 3, and the resultant absorbance of the ion-
pair extracted into the organic solvent measures the total concentration,
whereas at pH9 only the quaternary compound is ionised and forms
the extractable ion--pair.
The acid-dye technique is used for the assay of formulations containing
certain quaternary ammonium salts or amines, i.e... Biperidine Lactate
Injection, Clonidine Hydrochloride Injection and Tablets, Neostigmine
Methyl sulphate injection and Benzhexol Hydrochloride tablets.

Oxidation method:
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 propanol
amine, is the corresponding benzaldhyde derivative which exhibits
intense absorption, at around 240 nm owing to the interaction of the
carbonyl 𝜋 electrons with the ring electrons.

The assay of Ephedrine Hydrochloride Elixir involves the extraction of the

benzaldhyde into cyclohexane and measurement of the absorbance at its
λmax 241nm.
Compounds with a diphenylmethylidene [(C6H5)2C<] nucleus
are oxidized to benzophenone which has a λmax in hexane solution at
247nm.

Metal-ligand complexation:
Many organic reagents form complexes with metal atoms by the
formation of coordinate bonds and covalent bonds. Ligands with two or more
donating groups may share more than one pair of electrons with a single
metal atom by coordinating to two or more positions. The chelates formed by
these multidentate ligands with metal atoms are often coloured and
consequently their concentration may be determines by visible
spectrophotometry. Many examples of the photometric assay of either the
concentration of heavy metal ions by the addition of an excess of a chelating
agent or the concentration of organic substances by the addition of an excess
of a suitable complexing metal have been described.
 Characteristic colours which vary in hue with change of pH are given
by the reaction of iron (II) ions with phenols that contain two adjacent
hydroxyl groups. Thus, on mixing a solution of adrenaline with a
buffered solution containing iron(II) sulphate, a purple complex (λmax-
540 nm) is formed that has a maximum intensity at pH 8-8.5.

Assays of adrenaline in Procaine and Adrenaline injection and in

Lignocaine and Adrenaline injection of Methyldopa Tablets, Methyl dopate
injection and Isoprenaline Hydrochloride Injection are based upon the
coloured complexes formed with iron (II) sulphate-citrate reagent,
buffered with glycine buffer solution. Alternatively, the concentration of
iron(III) ions in a simple may be determined using the chromogenic
reagent Tiron (1,2-dihydroxy-benzene-3,5-disulphonic acid, disodium
salt), which forms an intense red complex.
One of the first substances to be used as a general reagent for the
photometric assay of heavy metal ions such as Cd, Hg, Cu, Pb and Zn was
dithizone(1,5-diphenylthiocarbazone).The coloured complex formed, for
example with lead:

is soluble in organic solvents and may be extracted from aqueous

solution into an immiscible organic solvent such as chloroform or carbon
tetrachloride. Lead dithizonate bas a carmine red colour in carbon
tetrachloride and the high absorptivity (€ 520 = 7 x 104) permits the
assay of only a few p.g., of lead. Thio-Michler's reagent [4,4'-bis­
(dimethyl amino)-thiobenzophenone] is an extremely sensitive chromo­
genic reagent for Pd ( €520 = 2.1 x 105) and for Hg (€560 = l.2 x10 5 ).

Determination of mimosine by a sensitive indirect

spectrophotometric method (diazotisation)

A simple and sensitive indirect spectrophotometric method is described

for the determination of mimosine based on its reaction with diazotized
sulfanilamide (DZSAM). DZSAM couples with N-(1-naphthyl)
ethylenediamine (NEDA) forming a pink colored azodye, absorbing
maximally at 540 nm (£max= 27 mM−1 cm−1). In the present method,
mimosine was first reacted with known excess of DZSAM and the
unreacted DZSAM was determined by coupling with NEDA. The reaction
of mimosine with DZSAM proceeded optimally at neutral pH. The decrease
in absorbance of the DZSAM–NEDA-coupled product obeyed Beer’s law in
the concentration range of 0.005–0.15 µgml−1 of mimosine. The present
method was applied to estimate mimosine in plant extracts containing
lesser than 0.05µgml−1 with recovery at 99±0.41%. The method
described is superior to other reported methods in terms of ease of
adaptability and sensitivity.
Effect of pH on the reaction of mimosine with DZSAM. Mimosine reacted
with DZSAM at varying pH: (1) pH 6.0, (2) pH 6.5, (3) pH 7.0, (4) pH 7.5
and (5) pH 8.0. Absorbance of the azodye formed plotted against the
concentration of mimosine.

Calibration graph for the determination of mimosine. Changes in the

absorbance of the azodye calculated as absorbance, A0 (when no
mimosine was added) absorbance, Amin, with added mimosine was plotted
against the corresponding concentration of added of mimosine. Standard
deviation in absorbance <0.001 (n = 6).

Colorimetric determination of steroids:

(Condensation)
Methods of colorimetric determination of ketosteroids which
make rise of the-reaction of a carbonyl group with a hydrazine, a
hydrazone or a hydrazide. Three of these methods use p-nitro- or 2,4-
dinitrophenylhydrazine, thus allowing the determination of steroids
bearing a carbonyl group at position 3, 17 or 20. Isoniazid gives colored
hydrazones only with Δ4- or Δ1,4 -3-ketosteroids, whereas salicyloyl­
hydrazide allows the fluorimetric determination of 17-ketosteroids. Under
suitable conditions, phenylhydrazine gives a colored species only with 17,
21-dihydroxy-20-ketosteroids (Porter-Silber reaction). These compounds
can also be determined with 2-hydrazinobenzothiazole or with 3-
methylbenzothiazolin-2-one hydrazone in the presence of an oxidant.

Spectrophotometric determination of pefloxacin in

pharmaceutical preparations (metal ligand
complexation)
It has been established that the antibiotic pefloxacin (Abaktal)
methane­ sulphonate reacts with Fe(III) at pH 1.00-8.00 to form a water-
soluble complex with maximum absorbance at 360 nm. The
composition of the complex, determined spectrophotometrically by
the application of Job's, molar-ratio and Bent-French's methods, was
pefloxacin: Fe(III) = 1:1 (pH= 2.50; λ= 360 nm; µ = 0.1 M). The relative
stability constant, obtained by the methods of Sommer and Asmus was
105(pH = 2.50; λ= 360 nm;. µ= 0.1 M). The molar absorptivity of the
complex at 360 nm was found to be 4.8 x 103 1 mol- em-\ Beer's law
was followed for pefloxacin concentrations of 2.15-85.88 J.g ml- 1The
lower sensitivity limit of the method was 2.15 µg ml-1 The relative
standard deviation (n = 10) was 0.57-1.07%. The method can be applied
to the rapid and simple determination of pefloxacin in aqueous solutions
and tablets
INDIRECT SPECTROPHOTOMETRIC DETERMINATION OF PIROXICAM
AND TENOXICAM THROUGH OXIDATION WITH POTASSIUM
PERMANGANATE (oxidation)
Three rapid, simple, accurate and selective validated spectrophotometric
methods (A, B and C) for the determination of piroxicam (PX) and
tenoxicam (TX) in bulk sample and in dosage forms are described. The
methods are based on the oxidation of the studied drugs by a known
excess of potassium permanganate in sulfuric acid medium and
subsequent determination of unreacted oxidant by reacting it with
Methylene Blue (Basic Blue 9) dye (method A), Acid Red 27 (Amaranth)
dye (method B) and Acid Orange 7 (orange II) dye (method C), in the same
medium at a suitable λmax = 660, 520 and 485 nm, respectively. The
reacted oxidant was found to be corresponding to the drug content.
Regression analysis of Beer-Lambert plots showed good correlations in
the concentration ranges 1.0-8.0, 1.0-9.0 and 1.0-7.2 µ g mL-1 using
methods A, B and C, respectively, for PX and 0.3-7.0, 0.3-1.6 and 0.3-2.5 µ g
mL-1 using methods A, B and C, respectively, for TX. The stoichiometric
ratios for the cited drugs to oxidant were studied. The optimum reaction
conditions and other analytical parameters were evaluated. The proposed
methods were applied successfully to determine the examined drugs
either in pure form or pharmaceutical formulations with good accuracy
and precision. The relative standard deviations were ≤ 0.33 with
recoveries 98.9-101.7% for PX and ≤ 0.49 with recoveries 99.4-102.0%
for TX.
Estimation of clonidine hydrochloride in pharmaceutical
preparations (Acid Dye Method):
An acid-dye complexing method with bromophenol blue, bromocresol purple and
methyl-orange was used for the ion-pa.ir extraction and colorimetric
determination of clonidine hydrochloride in pharmaceuticals containing 100 mg
of clonidine hydrochloride was 98.9% and relative standard deviation 0.89%.

REFERENCE:

1. Practical pharmaceutical chemistry by A.H.Beckett and

J.B.Stenlake; 4th edition; page no.: 300-306.
2. Elementary organic spectroscopy by Y.R.Sharma; page no.: 18-20.
3. www.sciencedirect.com
4. www.pubs.ac.in