Expt.

4
Post-Lab Discussion

Determination of Aspirin by
Spectrophotometry
Background
What is spectroscopy ?

Spectroscopy deals with the production, measurement,

and interpretation of spectra arising from the
interaction of electromagnetic radiation with
matter.

Note: There are different spectroscopic methods – to be discussed in

lecture.
UV-Vis Spectroscopy: spectroscopy utilizing radiation in the UV–Vis
range

 Electromagnetic radiation in the UV–Vis portion of the spectrum

ranges in wavelength from ~ 200 to ~ 700 nm.
(Note: In some literature, the wavelength range for UV-Vis may be somewhat higher or
lower, hence the symbol ~)

 The UV range runs from 200 to 350 nm; the Vis range from 350
to 700nm

 The UV range is colorless to the human eye.

 Different wavelengths in the visible range each have a characteristic

color.
UV-Vis spectroscopy may be divided into two general categories based
on the type of radiation–matter interaction that is being monitored. :

1. absorbance
2. fluorescence

Each of these two types of spectroscopy may be subdivided further

into qualitative and quantitative techniques.

In general, quantitative absorption spectroscopy is the most

common whose objective is to determine the concentration of
analyte in a given sample solution.
aComplementary hue refers to the color observed for a solution that shows
maximum absorbance at the designated wavelength assuming a
continuous spectrum “white” light source.
It is possible to predict which
wavelengths are likely to be
absorbed by a colored
substance.

When white light passes

through or is reflected by a
colored substance, a
characteristic portion of the
mixed wavelengths is
absorbed.

The remaining light will then

assume the complementary
color to the wavelength(s)
absorbed.
UV-Vis analysis is fast, simple and inexpensive method to
determine the concentration of an analyte in solution, where
the type of compound to be analyzed (‘analyte’) is known.
What can we analyze with UV-Vis analysis?

UV-Vis analysis is suitable for:

 analytes that can be dissolved in solvents like water, ethanol and hexane
 analytes that absorb UV or visible light

With UV/Vis quantitative measurements can be done on a single analyte in

solution (or more than one analyte in solution provided they do not interfere
with each other.)

Unsuitable are:
 analytes that have a photochemical reaction at (or above) the wavelength
range of interest
 unclear or colloidal samples
 Instrument used: Spectrophotometer

Sample models of spectrophotometers

Note: The wavelength region covered by a regular UV-Vis spectrophotometer is 200-1100 nm ( 1 nm, 10-9 m) from the
ultraviolet (UV: 200-380nm), the visible (380-750 nm), into the near infra red (IR: above 750 nm).
 A schematic diagram of a UV-visible spectrometer
 A combination of two lamps is used - a deuterium lamp for the UV part of the spectrum, and a
tungsten /halogen lamp for the visible part.
 A reference cell is one that, in theory, exactly matches the sample absorption cell with the exception
that it contains no analyte.
• A sample-holding cell or cuvette should be chosen after the general
spectral region to be used in a spectrophotometric measurement has
been determined.

• Sample holding cells vary in composition and dimensions. The sample

holding cell should be composed of a material that does not absorb
radiation in the spectral region being used.

• Cells meeting this requirement for measurements in the UV range may

be composed of quartz or fused silica.

• For the Vis range, cells made of silicate glass are appropriate and
inexpensive plastic cells also are available for some applications.
 The solution to be analyzed is contained in an absorption cell and placed in the
path of radiation of a selected wavelength(s)
 The amount of radiation passing through the sample is then measured relative to
a reference sample.
 The relative amount of light passing through the sample is then used to determine
the analyte concentration.

I0 = radiation coming in,

I = radiation coming out

Decrease of a beam of radiation as it passes through a cuvette containing an absorbing solution.

The decrease in radiant power as the beam passes through the solution is due to the capture (absorption) of
photons by the absorbing species.
 I0 I0 I

Factors contributing to the attenuation of a beam of radiation as it passes through a cuvette

containing an absorbing solution.
 Transmittance vs. Absorbance
The relationship between the power of the incident and exiting beams typically is
expressed in terms transmittance or the absorbance of the solution.

Transmittance (T) expresses the fraction of the
incident light absorbed by the solution, defined as the
ratio of I to I0.
T = I/I0
Transmittance also may be expressed as %.
%T = (I/I0) × 100
where:
T = transmittance
I0 = radiant power of beam incident on absorption cell
I = radiant power of beam exiting the absorption cell
%T = percent transmittance
Note: However, T and %T are not directly proportional to the
concentration of the absorbing analyte
Absorbance (A) is a measure of the
amount of light absorbed.
Absorbance is defined with respect to T.
A = log(I0/I) = − logT = 2− log %T
where:
A = absorbance
T and %T = as transmittance and
percent transmittance, respectively
Note: Absorbance is a convenient expression in
that, under appropriate conditions, it is directly
proportional to the concentration of the absorbing
species in the solution.
 The Beer-Lambert Law (or Beer's law)
According to the Beer-Lambert Law the absorbance is proportional to the
concentration of the substance in solution.

The Beer-Lambert Law can be expressed as:

A = εbc
where
A = absorbance
c = concentration of solution (M)
b = optical path length, i.e. dimension of the cuvette (cm)
ε = molar absorptivity (L/mol·cm), which is constant for a particular substance at
a particular wavelength; ↑ ε, ↑ amt. of radiation absorbed

Note: It should never be assumed that Beer’s law is strictly obeyed. There are several reasons for
which the predicted linear relationship between absorbance and concentration may not be observed.
In general, Beer’s law is applicable only to dilute solutions, up to approximately 10mM for most
analytes.
Limitations of the Beer-Lambert law
The linearity of the Beer-Lambert law is limited by chemical and
instrumental factors.

Causes of nonlinearity include:

 deviations in absorptivity coefficients at high concentrations
(>0.01M) due to electrostatic interactions between molecules in close
proximity
 scattering of light due to particulates in the sample
 fluoresecence or phosphorescence of the sample
 changes in refractive index at high analyte concentration
 shifts in chemical equilibria as a function of concentration
 non-monochromatic radiation, deviations can be minimized by using a
relatively flat part of the absorption spectrum such as the maximum of
an absorption band
 stray light
 errors in solution preparation may also be a contributing factor
The calibration curve
For quantitative measurements, it is advisable to use calibration curves in
most instances.

 The calibration curve is used to establish the relationship between analyte

concentration and absorbance.

 This relationship is established experimentally by analyzing a series of samples of

known analyte concentration (standards).

 The calibration curve is constructed by plotting the absorbance (y-axis) against

the corresponding concentrations (x-axis). It should be linear if the Beer-Lambert
Law is obeyed.

 A calibration curve can be used to determine the concentration of unknown

sample solution by measuring its absorbance.
Additional notes in preparing standard solutions:

 The standard solutions are best prepared with the same reagents
and at the same time as the unknown.

 The concentration range covered by the standard solutions must

include that expected for the unknown – very important for accurate
measurement.
A calibration curve
 The Chemical Reaction Involved
The ff. equation shows how acetylsalicylic acid is readily hydrolyzed in basic medium
to yield the salicylate dianion.

Salicylate dianion

The salicylate ion can be derivatized with Fe(III) to produce a violet colored
complex that can be quantified at its absorption maxima of 530nm.

Salicylate dianion + Iron (III) Tetraaquosalicylatroiron (III) or Fe-salicylate complex

Equations:

3C7H5O3- + Fe3+  Fe(C7H5O3)3

colorless violet complex
Data Processing
 Preparation of Calibration Curve

Concentration of Stock Solution 1.484 x 10-3 M

Concentration (M) Absorbance

blank 0
standard 1 0.073
standard 2 0.136
standard 3 0.232
standard 4 0.346
standard 5 0.451
 Preparation of Calibration Curve

Concentration of Stock Solution 1.484 x 10-3 M

Concentration (M) Absorbance

blank 0 0
standard 1 5.936 x 10-5 0.073
standard 2 1.187 x 10-4 0.136
standard 3 1.781 x 10-4 0.232
standard 4 2.374 x 10-4 0.346
standard 5 2.968 x 10-4 0.451
 Preparation of Calibration Curve

Wavelength used: 530 nm

Equation of the line:
Pearson correlation coefficient:
 Preparation of Calibration Curve

Wavelength used: 530 nm

Equation of the line: y = 1525.86x - 0.0201
Pearson correlation coefficient:
 Preparation of Calibration Curve

Wavelength used: 530 nm

Equation of the line: y = 1525.86x - 0.0201
Pearson correlation coefficient: 0.9938
 Determination of Acetylsalicylic Acid
Concentration
Trial 1 Trial 2
Mass of aspirin tablet (g) 0.1180 0.1050
Absorbance 0.2580 0.2680
Concentration of Acetylsalicylic
Acid from the calibration curve (M)
Adjusted concentrationof
Acetylsalicylic Acid due to df
(Vflask/Vpipet)
Mass of Acetylsalicylic Acid (g)
% Acetylsalicylic Acid
Average % Acetylsalicylic Acid
 Determination of Acetylsalicylic Acid
Concentration
Trial 1 Trial 2
Mass of aspirin tablet (g) 0.1180 0.1052
Absorbance 0.2580 0.2680
Concentration of Acetylsalicylic 1.823x10-4 1.625x10-4
Acid from the calibration curve (M)
Adjusted concentrationof 2.279x10-3 2.031x10-3
Acetylsalicylic Acid due to df
(Vflask/Vpipet)
Mass of Acetylsalicylic Acid (g) 0.1026 0.09134
% Acetylsalicylic Acid 86.95 86.83
Average % Acetylsalicylic Acid 86.89