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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
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.
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.
A1=a1bC1………………………………… (1)
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:
Combining this equation with Equation 2, one obtains the absorbance of the
fixed amount of reference standard referred to the unknown sample:
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:
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
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.
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).
DIFFERENT METHODS:
Diazotisation & Coupling.
Condensation Reaction.
Reduction of tetrazolium salts.
Acid-dye method.
Oxidation method.
Metal-ligand complexation.
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
Table 1
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''
N CONHN
N CONHNH3 A
O
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.
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.
REFERENCE: