Spectrophotometry and Colorimetry
Color comparison was one of the oldest methods used for quantitative
estimation of elements and substances. The variation of a color of a system with change
in concentration of some components forms the basis of colorimetric analysis. The color
is usually due to the formation of a colored compound by the addition of an appropriate
reagent at specific condition or it may be inherent in the desired constituent itself. The
intensity of the color may then be compared with that obtained by treating a known
amount of the substance in the same manner. Colorimetry is concerned with the
determination of the concentration of a substance by measurement of the relative
absorption of light with respect to a known concentration of a substance. In visual
colorimetry, natural or artificial white light (the whole range of the visible radiation is
from 400-760 nm) is generally used as a light source and determinations are usually
made with a simple instrument termed as colorimeter or color comparator. When the
eye is replaced by a photoelectric cell which eliminates the personal errors of the
observer is called a photoelectric colorimeter. When light of a limited spectral region,
say, violet, blue, green or yellow etc. is used in this instrument with the help of filters,
the instrument is then known as filter photometer. The filters may be of colored glass or
colored transparent polythene or plastic. Nowadays the filter has been replaced by a
monochromator, like prism or grating which controls the wavelength of light to the
maximum possible extent, the band-width should not exceed 5 Å– the instrument has
been named a spectrophotometer.
► Theory of spectrophotometry and colorimetry: When light (monochromatic or
heterogeneous) is incident upon a homogeneous medium, a part of the incident light is
reflected, a part is absorbed by the medium, and the remainder is transmitted as such. If
I0 denotes the intensity of the incident light, I t, the transmitted light, Ir the reflected light
and Ia the absorbed light, then it may be written:
I0 = Ia + It + Ir
If comparison cell of good quality is used, the value of Ir is very small and it may be
neglected. Under this condition, I0 = Ia + It
Bouguer actually investigated the range of absorption of light with the thickness of the
medium and Lambert extended this concept in quantitative field with solids. Beer later
applied Lambert’s concepts to solutions of different concentrations. Two separate laws
governing absorption generally known as Lambert’s and Beer’s law.
Dr. Jishnunil Chakraborty – Assistant Professor – St. Paul’s C. M. College, Kolkata- 700 009 1
Lambert’s law: When a monochromatic light passes through a transparent medium, the
rate of decrease in intensity (I) with thickness (l) of the medium is proportional to the
intensity of light.
Mathematically, –dI/dl I
On deduction it stands to It = I0 × e–kl (k is a proportionality constant)
On changing the above equation from natural to common logarithm,
It = I0 × 10–0.4343kl
or It = I0 × 10–Kl (K is called absorption coefficient)
The absorption coefficient is generally defined as the reciprocal of the thickness (l cm.)
required to reduce 1/10th of its intensity.
It/I0 = 0.1 = 10–Kl or K × l = 1 or K = 1/l
So unit of K is cm–1
The ratio (It/I0) is termed as transmittance (T) and the ratio (I0/It) is termed as opacity.
log(I0/It) is termed as the absorbance (A) of the medium or optical density (OD).
Beer’s law: When a beam of monochromatic light is allowed to pass through a solution,
the intensity of the light beam decreases exponentially with the increase in
concentration as well as thickness of the solution.
The equation becomes: It = I0 × 10–a.c.l (a is called molar absorption coefficient)
The value of ‘a’ depends on the unit of concentration. If c is expressed in gm-moles/litre,
l in cm, then a is replaced by ε and is termed as molar extinction coefficient.
The equation becomes: It = I0 × 10–ε.c.l
A = ε.c.l
or, ε = A/c.l
The unit of ε thus becomes litre gm-moles–1cm–1
Absorbance per unit path length (l) per unit concentration (c) is known as specific
extinction coefficient.
►Deviation from Beer’s law: From Beer’s law it follows that if we plot absorbance (A)
against concentration (c), a straight line passing through the origin should be obtained.
But sometimes a deviation from linearity is observed. Deviation from Beer’s law can
arise due to the following factors:
(i) It will hold over a wide range of concentration provided the structure of the
colored compound or the color of the solution does not change with change in
concentration, e.g.
Dr. Jishnunil Chakraborty – Assistant Professor – St. Paul’s C. M. College, Kolkata- 700 009 2
Cr2O72–(orange) + H2O 2CrO42–(yellow) + 2H+
(ii) Foreign substances or impurities present in the solution do not react with the
colored components or do not absorb or fluoresce any light.
(iii) Association (polymerization) or dissociation of the colored component
should not take place.
(iv) If monochromatic light is not used or if the slit width is not controlled,
undesirable radiations fall on the detector.
(v) It the solution is turbid- deviation occurs. This can be balanced using
matchable standard solutions.
►Application of colorimetry and spectrophotometry:
(i) Determination of molar composition of complexes.
(ii) Determination of instability constants of complexes.
(iii) Determination of pK value of an indicator.
(iv) Elucidation of structures of organic as well as inorganic compounds.
(v) Determination of molecular weight of colored substances.
►Instrumentation: A spectrophotometer is an instrument which resolves
polychromatic radiations into different wavelengths. A block diagram of a
spectrophotometer has been shown in the following figure. All spectrophotometers
require (i) a source of continuous radiation over the wavelengths of interest, (ii) a
monochromator for selecting a narrow band of wavelengths from the source spectrum,
(iii) a detector for converting radiant energy into electrical energy, and (iv) a device to
read out the response of the detector.
Optical part
Source Monochromator Sample Detector
Electrical part
Amplifier
Read-out
Figure 1: Block diagram of a spectrophotometer.
Dr. Jishnunil Chakraborty – Assistant Professor – St. Paul’s C. M. College, Kolkata- 700 009 3
►Spectrophotometric titrations:
Titrations without indicators: In a spectrophotometric titration the end point is
evaluated from data on the absorbance of the solution. For monochromatic light passing
through a solution, Beer’s law may be written as:
A = log(I0/It) = ε.c.l
Where I0 is the intensity of the incident light, It that of the transmitted light, ε is the
molar absorption coefficient, c is the concentration of the absorbing species, and l is the
thickness of or length of the light path through the absorbing medium. Since
spectrophotometric titrations are carried out in a vessel for which the light path is
constant, the absorbance is proportional to the concentration of the solution. Thus in a
titration in which the titrant, the reactant, or the product absorb radiation, the plot of
absorbance vs. the volume of titrant added will consist, if the reaction is complete and
the volume change is small, of two straight lines intersecting at the end point.
The shape of a photometric titration curve will depend upon the optical
properties of the reactant, titrant and product of the reaction at the wavelength used.
Some typical titration plots are given in Figure 2.
A B C D
Absorbance
Volume of titrant (ml.)
Figure 2: Different spectrophotometric titration plots.
Diagram A is characteristic of systems where the substance titrated is converted into a
non-absorbing product.
Diagram B is typical of the titration where the titrant alone absorbs.
Diagram C corresponds to systems where the substance titrated and the titrant are
colorless and the product alone absorbs.
Diagram D is obtained when a colored reactant is converted into a colorless product by
a colored titrant.
Titrations with indicators: In cases where none of the species involved in the titration
reaction absorbs sufficiently, an indicator may be added to the solution. Figure 3 shows
an example of an indicator titration, a chelometric titration of Cu 2+ ion with EDTA using
Dr. Jishnunil Chakraborty – Assistant Professor – St. Paul’s C. M. College, Kolkata- 700 009 4
the metallochromic indicator pyrocatechol violet. In this titration, a wavelength is
selected where the free indicator absorbs more strongly than the copper-indicator
complex. We see in the figure first the reaction of free Cu2+ ion with EDTA, which does
not affect the absorbance of the solution at this wavelength. Then, as the end point is
approached, copper is pulled away from the indicator by the titrant, and the absorbance
rises as free indicator accumulates, until finally all of the copper has been titrated and
the absorbance becomes constant again.
Absorbance
0 1 2 3
ml. of EDTA solution
Figure 3: Titration of Cu2+ with EDTA using pyrocatechol violet indicator.
Titration of mixtures: Figure 4 shows a photometric titration of a mixture of bismuth
and copper with EDTA. At the wavelength selected, the Cu(II)-EDTA chelate absorbs
strongly, while the other species (Bi3+, Bi(III)-EDTA chelate and EDTA) have ε-values of
zero. The bismuth(III) chelate is much more stable than the copper(II) complex. Thus as
EDTA is added to the Bi3+-Cu2+ mixture, the bismuth(III) chelate is formed first. When
[Bi3+] has been reduced to a very low value, the Cu(II)-EDTA chelate begins to form, and
because this is the strongly absorbing species, the absorbance begins to rise. After the
copper end point, the curve levels off as excess, non-absorbing EDTA is added.
Absorbance
Cu end
point
Bi end
point
0 1 2 3 4 5
ml. of EDTA solution
Figure 4: Titration of Bi3+-Cu2+ mixture with EDTA.
Dr. Jishnunil Chakraborty – Assistant Professor – St. Paul’s C. M. College, Kolkata- 700 009 5
Owing to the linear response of absorbance to concentration, an appreciable
break will often be obtained in a photometric titration, even though the changes in
concentration are insufficient to give a clearly defined inflexion point in a photometric
titration. Photometric titrations have several advantages over direct colorimetric
determinations. The presence of other substances absorbing at the same wavelength
does not necessarily cause interference, since only the change in absorbance is
significant. The precision of locating the titration line (required for the evaluation of the
equivalence point) by pooling the information derived from several points is greater
than the precision of any single point; furthermore, the procedure may be useful for
reactions which tend to be appreciably near the equivalence point. An accuracy and
precision of a few tenths percent are attainable with comparative ease by
spectrophotometric titration. The optimum concentration of the solution to be analyzed
depends upon the molar absorption coefficient of the absorbing species involved, and is
usually of the order of 10–4 to 10–5 (M). The effect of dilution can be made negligible by
the use of a sufficiently concentrated titrant. If relatively large volumes of titrant are
added the effect of dilution may be corrected by multiplying the observed absorbances
by the factor (V + v)/V where V is the initial volume and v is the volume added; if the
dilution is of the few per cent the lines in the titration plots appear straight. The
operating wavelength is selected on the basis of two considerations: avoidance of
interference by other absorbing substances and need for an absorption coefficient
which will cause the change in absorbance to fall within a convenient range. The latter is
particularly important, because serious photometric error is possible in high-
absorbance regions. Light leakage must, of course, be avoided.
The experimental technique is simple. The cell containing the solution to be
titrated is placed in the light path of a spectrophotometer, a wavelength appropriate to
the particular titration is selected, and the absorption is adjusted to some convenient
value by means of the sensitivity and slit-width controls. A measured volume of the
titrant is added to the stirred solution, and the absorbance is read again. This is
repeated at several points before the end point and several more points after the end
point. The latter is found graphically.
►Spectrophotometric estimation of:
Iron: To the solution 10% hydroxylamine hydrochloride is added in order to reduce
Fe(III) to Fe(II). 1% 1,10-phenanthroline (o-phen) is added to form the [FeII(o-phen)3]2+
Dr. Jishnunil Chakraborty – Assistant Professor – St. Paul’s C. M. College, Kolkata- 700 009 6
complex. NaOAc/AcOH buffer is added to maintain pH =3.5. The absorbance is
measured at 510 nm. Series of standards are to be treated in the same way. The
concentration of Fe(II) in the given sample is obtained from the calibration plot.
Manganese: Small quantities of manganese are usually estimated colorimetrically by
oxidation to permanganic acid, using periodate as oxidant. In hot acid solution (HNO 3 or
H2SO4), periodate oxidizes manganese(II) quantitavely to HMnO4:
2Mn2+ + 5IO4– + 3H2O 2MnO4– + 5IO3– + 6H+
The merits of the periodate method include: (a) the concentration of the acid has little
influence, and may be varied within wide limits; (b) the may be prolonged beyond the
time necessary to oxidize Mn(II) without detriment; and (c) the permanganic acid
solution may be preserved for several months if an excess periodate is present.
Frequently ferric ion is added to the standard in amount equal to that found
independently to be present in the sample. H3PO4 must be present to prevent the
precipitation of ferric periodate and iodate, and also to decolorize the ferric ion (by
complex formation). If chloride ions are present, it is necessary to evaporate with a
mixture of HNO3 and H2SO4 until fumes of the latter appear. Chloride reacts with
periodate. Reducing substances reacting with periodate or permanganate must be
destroyed before the periodate oxidation. A wavelength of 545 nm should be used to
have maximum absorption for MnO4– and the concentration of Mn(II) in the sample is
determined from the calibration plot.
Phosphorus: Phosphorus is estimated as orthophosphate by molybdenum blue
method. Orthophosphate and molybdate ions condense in acidic solution to give
molybdophosphoric acid (phosphomolybdic acid), which upon selective reduction (with
hydrazinium sulfate) produces a blue color due to molybdenum blue of uncertain
composition. The intensity of the blue color is proportional to the amount of phosphate
initially incorporated in the heteropoly acid. If the acidity at the time of reduction is 0.5
(M) in H2SO4, and hydrazinium sulfate is the reductant, the resulting blue complex
exhibits maximum absorption at 820-830 nm.
Dr. Jishnunil Chakraborty – Assistant Professor – St. Paul’s C. M. College, Kolkata- 700 009 7
