FA3111

INSTRUMENTAL PHARMACEUTICAL
ANALYSIS

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Daryono Hadi Tjahjono
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Sophi Damayanti
Elin Julianti
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Benny Permana
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Fransiska Kurniawan
Tasia Amelia
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Winni Nur Auli

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SCHOOL OF PHARMACY
INSTITUT TEKNOLOGI BANDUNG
2020
 TABLE OF CONTENTS

TABLE OF CONTENTS ..................................................................................................................1

INFRARED SPECTROSCOPY ......................................................................................................2

ULTRAVIOLET AND VISIBLE ABSORPTION SPECTROPHOTOMETRY ............................6

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ........................................................ 10

GAS CHROMATOGRAPHY ........................................................................................................ 15

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FLAME SPECTROSCOPY .......................................................................................................... 23

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SPECTROFLUOROMETRY ........................................................................................................ 28

POTENTIOMETRY ....................................................................................................................... 32

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BIAMPEROMETRY....................................................................................................................... 41

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1
 INFRARED SPECTROSCOPY

1. INTRODUCTION
Infrared spectroscopy is a technique based on the vibrations of the atoms of a molecule.
An infrared spectrum is commonly obtained by passing infrared radiation through a
sample and determining what fraction of the incident radiation is absorbed at a particular
energy. The energy at which any peak in an absorption spectrum appears corresponds
to the frequency of a vibration of a part of a sample molecule. IR spectroscopy provides
a way of finding functional groups present in a molecule because it detects the stretching
and bending of chemical bonds. For a molecule to show infrared absorptions it must

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possess a specific feature, i.e. an electric dipole moment of the molecule must change

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during the vibration. This is the selection rule for infrared spectroscopy.

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The interactions of infrared radiation with matter may cause changes in molecular dipoles
associated with vibrations and rotations. In order to begin with a basic model, a molecule
can be looked upon as a system of masses joined by bonds with spring-like properties.

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Taking first the simple case of diatomic molecules, such molecules have three degrees

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of translational freedom and two degrees of rotational freedom. The atoms in the
molecules can also move relative to one other, that is, bond lengths can vary or one atom
can move out of its present plane. This is a description of stretching and bending
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movements that are collectively referred to as vibrations. For a diatomic molecule, only
one vibration that corresponds to the stretching and compression of the bond is possible.
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This accounts for one degree of vibrational freedom. Polyatomic molecules containing
many (N) atoms will have 3N degrees of freedom. Looking first at the case of molecules
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containing three atoms, two groups of triatomic molecules may be distinguished, i.e.
linear and non-linear.
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Figure 1.1. Types of molecular vibrations

Analytical infrared (IR) spectroscopy includes several methods that are based on the
absorption (or reflection) of electromagnetic radiation with wavelengths in the range

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 of 1 to 1000 µm. The spectral range can be divided into three groups: near IR (1 to
2.5 µm), mid IR (2.5 to 25 µm) and far IR (beyond 25 µm). Although the near-IR is
poor in specific absorptions, it is considered as an important method by quality control
laboratories for quantitative applications. By contrast the mid-IR region provides more
information upon the structures of compounds and consequently it is much used as
a procedure for identifying organic compounds for which it remains a form of
functional group fingerprinting. The far IR requires the use of specialized optical
materials and sources. All infrared spectrometers generate the infrared spectrum.

Structure compound can be interpreted by IR spectrum, especially the identification

of functional groups.

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Figure 1.2. Important IR Absorption

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1.1 Infrared instruments

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There are three types of instruments for infrared measurements are available:
1) Dispersive spectrometers
2) Non-dispersive IR photometers
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3) Fourier transform infrared spectrometer (FTIR)

The IR spectrum has the wavenumbers range from 4000 to 600 or 400 cm -1. Fourier
transform (FT) IR spectrometers offer a number of advantages over the older IR
spectrometers. Since the data are collected and stored in a digitized form in the computer,
they can be easily manipulated, transported, and displayed. Spectra can be added or
subtracted, and this technique is useful for subtracting the solvent spectrum from a
spectrum of a sample in that solvent. FT IR instruments are faster, more sensitive, and
more accurate when compared to the older spectrometers. In an FTIR spectrometer, all
of the infrared frequencies are measured simultaneously instead of individually, case in

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 older IR instruments. A schematic diagram of an FT IR spectrometer is depicted in Fig.
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Figure 1.3. Schematic of an FT IR spectrometer
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1.2 Sample analysis technique
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Gas sample
Cells with a minimum optical path of a few centimeters are used for gas analysis. If the
absorbance under these conditions is too weak, a cell using mirrors to increase the path
length can be used.
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Liquid sample
Liquids are usually analyzed with cells which have dismountable IR windows. For
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qualitative analysis, a droplet of the sample is compressed between two NaCl or KBr
disks without a divider.
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However, for quantitative analysis, either InfrasilTM quartz cells (with an optical path from
1 to 5 cm) or cells that have a variable or fixed width, generally smaller than 1 mm, can
be used. In the mid-infrared, the latter consist of two KBr or NaCl windows with a spacer.
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The optical path length must be calibrated and periodically controlled.

Solid sample
For solids, many approaches can be used:
- A few milligrams of sample may be dissolved in paraffin oil (NUJOL), which has only
three interfering bands.
- The sample is crushed along with dry KBr in a mortar. The mixture is then compressed
into a disc using a hydraulic press with pressure 5 to 8 t/cm2 or manually. The resulting
translucent fritted disc corresponds to a dispersion of the sample in a solid matrix.
Diffusion losses of the radiation can be reduced when the solid is crushed into a fine
powder. Not all solids are compatible with this crushing technique.
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 - If the solid can be dissolved in a suitable solvent, then the same method as for liquids
can be used.

2. APPARATUS
Infrared Spectrometer Jasco FT/IR-4200, micro KBr pellet, KBr pellet holder, hydraulic
press, mortar, stamper, spatula.

3. CHEMICALS
Freshly-dried spectra-grade KBr and dried pure organic compounds.

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4. GENERAL PROCEDURE
KBr pellet method is employed in this experiment:

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1. Grind vigorously a little amount of dry sample on a clean and dry agate mortar.

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2. Add about freshly-dried spectra-grade KBr (approximately 1:100), and grind well
to a fine homogenous powder.
3. Take two stainless steel disks (micro KBr pellet template), fill the cutout hole with

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the finely ground mixture.

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4. Put the second stainless steel disk on top and transfer the sandwich onto the
hydraulic press.
5. With a pumping movement, move the hydraulic pump handle downward. Leave
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for a few seconds, and release the pressure.
6. Remove the disks and pull apart. The finished KBr pellet must be transparent to
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visible light.
7. Insert the KBr pellet containing the sample on the KBr pellet holder.
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8. Develop the IR spectra of the sample.

5. REFERENCES
Rouessac, F. and A. Rouessac, 2005, Chemical Analysis; Modern Instrumentation
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Methods and Techniques, John Wiley & Sons, London, 161-186.

Skoog, D.A, F.J. Holler, and T.A. Nieman, 1998, Principles of Instrumental Analysis, 5th
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ed., Harcourt College Publ., Orlando, 381-426.

Shriner, R. L., C.K.F. Hermann, T.C. Morrill, D.Y. Curtin, and R.C. Fuson, 2004, The
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systematic Identification of Organic Compounds, 8th ed., John Wiley & Sons,
Singapore, 194-200.
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Watson, G., 2001, Pharmaceutical Analysis, Churchill Livingstone, London, 97-110.

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 ULTRAVIOLET AND VISIBLE ABSORPTION SPECTROPHOTOMETRY

1. INTRODUCTION
Spectroscopy is the study of light as a function of wavelength that has been emitted,
reflected or scattered from a solid, liquid, or gas. Spectrometry is derived from
spectrophotometry, the measure of photons as a function of wavelength, a term used for
years in astronomy. Light or electromagnetic radiation may be regarded as being
wavelike or considered as a stream of energy packets or particles traveling with a high
velocity. UV/Vis absorption spectrophotometry is the term used when the radiation of

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ultraviolet and visible region absorbed by molecules is measured.

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1.1 Instrumentation
Ultraviolet and visible spectrophotometer consists of (1) sources, (2) wavelength
selectors, (3) sample containers, (4) radiation transducers, and (5) signal processors and
readout devices.

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Figure 2.1. Single beam spectrometer

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In common with the other optical elements of an absorption instrument, the cells, or
cuvettes that hold the sample and solvent must be constructed of a material that passes
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radiation in the spectral region of interest. Quartz or fused silica is required for work in
the ultraviolet region (below 350 nm); both of these substances are transparent in the
visible region. Silicate glasses can be employed in the region between 350 and 2000 nm.
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Plastic containers have also found application in the visible region. The most common
cell length for studies in the ultraviolet and visible regions is 1 cm.

1.2 Analytical Spectrometry

The set of energy levels associated with a particular substance is a unique characteristic
of that substance and determines the frequencies at which electromagnetic radiation can
be absorbed or emitted. Qualitative information regarding the composition and structure
of a sample is obtained through a study of the positions and relative intensities of
spectral lines or bands. Quantitative analysis is possible because of the direct
proportionality between the intensity of a particular line or band and the number of atoms
or molecules undergoing the transition.
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 Spectroscopic measurements for the UV, visible and infrared regions are most
conveniently and reliably made by determining the absorbance of a solution. The
absorbance tends to be a robust measure that is reproducible and only slightly affected
by common variables of temperature and trace impurities. The absorbance of a system
is determined by measuring the intensity of light at a given wavelength, I0 and then
measuring the intensity with a sample in the same beam of light, I. The ratio of these
intensities is the transmittance, T.
T = I/Io
When using a single-beam spectrometer, I0 is measured when a reagent blank is used

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to ‘‘zero’’ the absorbance scale. The value of I is then measured when the sample is
inserted into the spectrometer. On the other hand, when using a double-beam instrument

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both the reagent blank, I0, and the sample, I, are measured continuously and the

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appropriate ratio is determined electronically. The absorbance is defined as:
A = -log T
The Lambert-Beer Law relates the absorbance to concentration in two alternate forms

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depending on the units used for the concentration:

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A=abc or A=εbc
Modern terminology defines A as the absorbance, a as the absorptivity, b as the optical
path length and c as the concentration. In the latter equation ε represents the molar
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absorptivity.
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The Lambert-Beer Law assumes that the electromagnetic radiation being absorbed is
monochromatic. In practical instruments it is not a single wavelength but a band of
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wavelengths that enter the sample.

Table 2.1. Terminology and units used for the Lambert-Beer law
A a or ε B c
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A = abc Absorbance Absorptivity Optical path Concentration

dimensionless (g-1.cm-1) length (cm) (g L-1)
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A = εbc Absorbance Molar absorptivity Optical path Concentration

dimensionless (mol-1.cm-1) length (cm) (mol L-1)
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If the absorptivity or molar absorptivity are not approximately equal, the linear
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relationships of the Lambert-Beer Law will not hold.

In practical situations the absorbance of a sample is determined by making two

measurements, the first to determine I0 and the second to determine I. The determination
of Io is used to cancel a large number of experimental factors that could affect the result.
When measuring I0 the sample container (the cuvette) must closely match the unknown
container in all ways except for the analyte content.

Choice of solvent
A solvent to be used in colorimetric or spectrophotometric determinations must meet the

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 following requirements:
• must be a good solvent for the substance under determination,
• does not interact with the solute, and
• must not show significant absorption at the wavelength to be employed in the
determination.

For inorganic compounds water usually meets these requirements, but for the majority of
organic compounds it is necessary to use an organic solvent.

An immediate complication which arises is that polar solvents such as alcohols, esters

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and ketones (water also falls into this category) tend to obliterate the fine structure of
absorption spectra which are related to vibrational effects. To preserve these details, the

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absorption measurements must be carried out in a hydrocarbon (non-polar) solvent.

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Thus, for example, a solution of phenol in cyclohexane gives an absorption curve showing
three sharply defined peaks in the ultraviolet, but an aqueous solution of the same
concentration gives a single broad absorption band covering the same wavelength range

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as that observed in the hydrocarbon solvent.

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There is the further complication as far as spectrophotometry is concerned, i.e. that all
solvents show absorption at some point in the ultraviolet, and care must be taken to
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choose a solvent for a particular determination which is transparent in the requisite
wavelength region. Some organic solvents are listed below in order of 'cut-off
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wavelengths', which is the wavelength at which the transmittance is reduced to 25 %
when measured in a l.0 cm cell against water as reference material.
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Cut-off Wavelength Cut-off wavelength

Solvent Solvent
(nm) (nm)
Water 190 Cyclohexane 212
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Hexane 199 Dichloromethane 233

Heptane 200 Chloroform 247
Diethyl Ether 205 Carbon tetrachloride 257
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Ethanol 207 Benzene 280

Methanol 210 Acetone 331
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2. APPARATUS
UV/Vis spectrophotometer Beckman DU 605i, quartz cuvette, analytical balance,
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volumetric glassware, beaker glass, dropper pipette, spatula, wash bottle, lens paper.

3. GENERAL PROCEDURE
1. Prepare the following solutions:
a. Standard and sample solvent: depend on the compendial
b. Stock solution:
Dissolve 25 mg of active ingredient in its solvent in 25-mL glass volumetric
flask (concentration stock solution will be 1 mg/mL)
c. Standard solutions:
By using 10-mL volumetric flasks, prepare six standard solutions containing 2,
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 4, 6, 8, 10, and 12 μg/mL active ingredient from the stock solution, dilute the
solution with its solvent. All standard solutions are prepared in glass volumetric
flask.

Note: the observed absorbance should be in the range of 0.2 to 0.8 absorbance
units, the optimal range for reading absorbance values that follow the Lambert-
Beer equation. If the observed absorbance is out of this range, the sample
solutions have to be re-diluted to obtain appropriate concentrations.

2. Sample Analysis (General Procedure)

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a. Fill two-thirds of a quartz cuvette with standard solution of 10 μg/mL active
ingredient and place in to the ‘sample compartment’ of the

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spectrophotometer and another cuvette containing solvent in the ‘blank

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compartment’. Develop an absorption curve of active compound by using
one of the standard solutions with “Wavelength Scan” Procedure
b. Note the wavelength of absorbance peak

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c. Quit “Wavelength Scan” program

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d. Measure the absorbance of each standard and sample at the maximum
wavelength of paracetamol with Fixed Wavelength/Absorbance Reading
Procedure
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e. Make a calibration curve of absorbance versus concentration (the
concentration of standards [x-axis] versus absorbance [y-axis]).
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f. Use the equation of linear regression from a calibration graph to calculate
the active ingredient concentration of the sample.
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g. Report the concentration of your sample solution, active ingredient content

in your tablet and also the molar absorptivity (ε) of active ingredient.

4. REFERENCES
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Rouessac, F., A. Rouessac, 2005, Chemical Analysis; Modern Instrumentation

Methods and Technique, John Wiley & Sons, 189-198.
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Skoog, D.A, F.J. Holler, and T.A. Nieman, 1998, Principles of Instrumental Analysis,
5th ed., Harcourt College Publ., Orlando, 300-351.
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Watson, G., 2001, Pharmaceutical Analysis, Churchill Livingstone, London, 75-94

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 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

1. INTRODUCTION
Liquid chromatography refers to any chromatographic procedure in which the moving
phase is a liquid. Traditional column chromatography (whether adsorption, partition, or ion-
exchange), thin- layer and paper chromatography, and HPLC are each examples of liquid
chromatography. Chromatographic separation is the result of specific interactions between
sample molecules and the stationary and moving phases.

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High performance liquid chromatography is the most widely used of the analytical
separation techniques. The reasons for the popularity of the method is the sensitivity, ready

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adaptability to accurate quantitative determinations, suitability for separating non-volatile

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species or thermally fragile ones, and above all, its applicable to prime interest substance
in industry many fields of science, and public. The main disadvantages of preparative-scale
separation is the large amount of solvents. HPLC can analyze amino acid, proteins, nucleic

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acids, hydrocarbons, carbohydrates, drugs, terpenoids, pesticides, antibiotics, steroids,
metal-organic species, and a variety of inorganic substances.

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Figure 3.1. Schematic of an apparatus for HPLC.

Components of a HPLC instrument

1. Solvent system
The apparatus is equipped with one or more glass or steel solvent reservoirs. The solvent
may be heated or stirred, or the solvent reservoirs may contain attachments inlets for inert
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 gases for degassing. The presence of dissolved gases results in poorer resolution of the
peaks.

2. Pumps
HPLC system has one or several pumps. The pump will press the mobile phase through
the packing. The pump generates pressures up to 6000 psi to drive the mobile phase at a
flow rate of
0.1 – 10 mL/min. Through a long, narrow column of very fine particles. This elevated
pressure, created before the injector, depend on the flow rate, the viscosity of the mobile
phase and the size of the particles of stationary phase.

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A separation that employs a single solution of constant composition is termed as isocratic
elution. Frequently, separation efficiency is greatly enhanced by gradient elution. Two or

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three solvent system with significantly difference in polarity are employed. During the

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elution, the ratio of the solvents may be constant or varied in a programmed way (the
composition of the mobile phase varies with time).

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3. Sample introduction

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The solution of the analyte in a syringe is injected into a loop of small defined volume are
either integrated in the rotor of the valve or are connected to the outside of the valve.
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Figure 3.2. A sampling loop for liquid chromatography. With the valve handle as shown on
the left, the loop is filled from the syringe, and the mobile phase flows from
pump to column. When the valve is placed in the position on the right, the
loop is inserted between the pump and the column so that the mobile phase
sweeps the sample onto the column.

4. Columns
Columns for HPLC are usually constructed from stainless steel tubing. Fittings and plugs
must be inert and should not detract from the homogeneity of the flow. Analytical columns
range in length from 10 to 150 cm long. The inside diameters vary from 1 to 20 mm.
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 5. Stationary phases
A successful separation depends on the choice of the stationary phase, which is
characterized by several surface parameters.

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Figure 3.3. HPLC column and guard column, assembly and its exploded view. The
removable precolumn (Guard cartridge) which is not expensive, prevents clogging of the
analytical column while extending its lifetime and preserving its performance.

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Silica gel

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With the exception of a few organic polymers, silica gel represents the basic material is
used to pack HPLC columns. It is prepared under conditions of controlled hydrolysis by
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polymerization of tetraethoxysilane in the form of an emulsion giving rise to microspheres
of uniform diameter in the order of 2 to 5 μm. A sol-gel is formed in the process and these
very small particle must grow in a regular manner in order to obtain a diameter of a few
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micrometers. The material has to be free of metallic ions. The silica gel particles obtained
must be in uniform diameter to avoid the presence of preferential pathways in the packed
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bed in the column. Silica gel is a polar material. The mode of action of silica gel is based
on adsorption, a phenomenon that leads to the accumulation of a compound at the
interface between the stationary and mobile phases.
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Bonded Silica
The reaction of alkylchlorosilanes with silica gel in the presence of an alkaline agent
represents a typical transformation procedure. This reaction is used to obtain phases
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such as RP-8 (dimethyloctadecylsilane groups), RP-18 or ODS (dimethyloctadecylsilane

groups). This procedure converts approximately half of silanol groups into bonded
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groups. The reaction is more complete with chlorotrimethylsilane (ClSiMe3) or

hexamethyldisilisane (Me3SiNHSiMe3).
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6. Mobile Phases
The degree of interaction between the mobile phase and the stationary phase, whether
the latter be normal or reversed phase, affects the retention times of the analytes. In
principle, the polarity of the stationary phase can lead to two situations:
- if the stationary phase is more polar than mobile phase, the technique is called
normal phase chromatography.
- if the stationary phase is less polar than mobile phase used (most commonly
methanol or acetonitrile with water), the technique is called reversed phase
chromatography.

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 7. Detectors
The most widely used detectors for chromatography are based on the optical properties
of the analytes: absorption, fluorescence and refractive index.

Spectrophotometric detectors
Absorbance detector measure at the end of the column, the analyte absorption at one or
many wavelengths in the UV or Visible spectrum. In order to detect the compounds which
are eluted from the column, it is essential that the mobile phase be transparent or possess
a very small absorption.

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Spectrofluorometric detectors
Some compounds are fluorescent, in that they have the ability to re-emit part of the light

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absorbed from the excitation source. In practice, fluorescence is measured perpendicular

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(at °90) to the exciting radiation source. The intensity of the fluorescence is proportional
to the concentration of analyte, as long as this concentration is kept low. The application
of this very sensitive and selective detector can be extended by using derivatization,

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either pre- or post- column.

Refractive index detectors

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Schematically, a beam of light (mono- or polychromatic) travels through a cell that has
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two compartments. One compartment is filled with the pure mobile phase while the other
is filled with the mobile phase eluting from the column. A compound eluting from the
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column causes a change in refractive index which results in an angular displacement
(refraction) of the beam. In practice, the signal corresponds to a measure of retroaction
that must be provided to the optical element in order to compensate for the beam
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displacement. The detector can only be used in the isocratic mode because in gradient
elution the composition of the mobile phase changes with time, as does the refractive
index.
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Other detectors
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Electrochemical detectors (PAD), evaporate light scattering detector (ELSD), mass

spectrometric detectors (MS).
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2. APPARATUS
HPLC Hewlett Packard Series 1100, C-18 column, analytical balance, micro-syringe,
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volumetric glassware.

3. CHEMICALS
Caffeine, paracetamol, double distilled water, methanol pro HPLC.

4. PROCEDURES
A. Standards and Sample Preparation
a. Caffeine stock solution (1 mg/mL):
50 mg of caffeine in a 50-mL volumetric flask dilute to the mark with HPLC
solvent.

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 b. Paracetamol stock solution (1 mg/mL):
50 mg of paracetamol in a 50-mL volumetric flask dilute to the mark with
HPLC solvent.
c. Caffeine and paracetamol standards solution:
Prepare five standards solution containing caffeine and paracetamol with the
concentration 10, 20, 30, 40, 50, and 60 ppm from the caffeine and
paracetamol stock solution. Dilute the solutions with HPLC solvent.
d. Sample solution:
The sample solution will be given by the assistant.

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B. HPLC Determination of Caffeine and Paracetamol in The Sample

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1. Measure the standard and sample with the following HPLC system:

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- Column : C-18
- Detector : UV
- Wavelength : 272 nm

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- Flow rate : 0.8 ml/min
- Mobile phase : Double distilled water : methanol (60 : 40)

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2. Inject pure caffeine solution into the HPLC system, obtain a chromatogram
and record the retention time of caffeine.
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3. Inject pure paracetamol solution into the HPLC system, obtain a
chromatogram and record the retention time of paracetamol.
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4. Individually inject each standard, obtain a chromatogram for each one and
record the retention time and peak area of caffeine and paracetamol.
5. Inject the sample into the HPLC system; repeat 3 times.
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6. All of the standards and sample should be filtered through a 0.45 μm filter
before injected into the HPLC system.
7. Calculate the concentration.
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5. REFERENCES
Kazakevich Y and Lobrutto R., 2007, HPLC for pharmaceutical scientists., Wiley-
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Interscience,
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Rouessac, F. and A. Rouessac, 2005, Chemical Analysis: Modern Instrumentation

Methods and Techniques, John Wiley & Sons, London, 45-62.

Skoog, D.A, F.J. Holler, and T.A. Nieman, 1998, Principles of Instrumental Analysis,
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5th ed., Harcourt College Publ., Orlando, 725-766.

Watson, G., 2001, Pharmaceutical Analysis, Churchill Livingstone, London, 237-274.

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 GAS CHROMATOGRAPHY

1. INTRODUCTION
Gas chromatography (GC) is that form of chromatography in which a gas is the moving
phase. GC is an appropriate procedure for analyzing many compounds in a great variety
of applications, provided these compounds are volatile, thermally stable and with
reasonable polarities to avoid problems encountered with derivatization procedures.

Gases of low viscosity with favorable solute diffusivity, such as hydrogen and helium, are
commonly used as mobile phases in GC, the sample is vaporized and carried by the mobile

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gas phase through the column. Samples partition (equilibrate) into the stationary liquid
phase, based on their solubilities at the given temperature. The component of the sample

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(analytes) separate from one another based on their relative vapor pressures and affinities

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for the stationary bed. This type of chromatographic process is called elution.

In GC, the samples, or derivatives thereof, must be volatile at the column operating
temperature. Any substance, organic or inorganic, which exhibits a vapor pressure of at

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least 60 torr (the column temperature may be raised to 350°C) can be eluted from a GC

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column. The sample is vaporized and injected onto the head of a chromatographic column.
Elution is brought about by the flow of an inert gaseous mobile phase. In contrast to most
other types of chromatography, the mobile phase does not interact with molecules of the
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analyte; its only function is to transport the analyte through the column.
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Figure 1. Schematic representation of the chromatographic process

1.1 Components of a gas chromatograph

The basic components of a gas chromatograph are as follows
a. A supply of carrier gas with attendant pressure regulator and flow controller;
b. An injection port or valve and followed possibly by a splitter;

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c. A separation column;
d. A detector
e. A thermostatically controlled oven that is also able to be programmed for various
heating rates
f. A recorder or other readout device

The analysis starts when a sample in liquid or gaseous state is injected. The dual role of
the injector is to vaporize the analytes and to mix them uniformly in the mobile phase. Once
the samples is vaporized in mobile phase, it is swept into the column, which is usually a
tube coiled into a very small section with a length that can vary from 1 to over 100 m. The

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column containing the stationary phase is situated in a variable temperature oven. At the
end of the column, the mobile phase passes through a detector before it exits to the

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atmosphere. Some gas chromatograph models have their own power supply, permitting

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them to be used in the field.

Each of the principal modules is composed of several electronic and pneumatic

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subsystems.

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Figure 2. A schematic diagram of a gas chromatograph

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1.1.1 Carrier gas and flow regulation

The mobile phase is a gas (helium, nitrogen or hydrogen) which can be purchased from
industrial sources or generated on-site in the case of nitrogen and hydrogen since the
flows are relatively small (1 to 25 mL/min depending on column type). The carrier gas
should not contain traces of water or oxygen because these are very deleterious to
stationary phase. Typically, a filtering system containing a drying agent and a reducing
agent is used between the gas source and chromatograph.

The carrier gas is chosen for its inertness. Its only purpose is to transport the analyte
vapors through the chromatographic system without interaction with the sample
components. The gas is obtained from a high-pressure gas cylinder and should be free
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from oxygen and moisture. Premium grades of carrier gas are usually less expensive in
the long run. Helium is the most popular, but it is expensive. Hydrogen is increasing in
popularity, sometimes the choice of carrier gas is dictated by the detector.

Traditionally, chromatographers have used helium and nitrogen as the carrier gases of
choice in gas chromatography. A laboratory-size generator produces ultrapure and ultra-
dry (99.999%) hydrogen gas by the electrolysis of deionized or distilled water and
separation of hydrogen from other electrolysis products by permeation through a
palladium membrane. A replaceable desiccant cartridge reduces water in the generated
hydrogen to less than 80 ppm. Gaseous hydrogen can be stored safely and conveniently

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using solid metal hydrides; the hydrogen is stored at the pressure of a comparable
cylinder. With adequate equipment, hydrogen poses no greater hazard in the laboratory

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than any other compressed gas.

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Hydrogen and helium always permit faster analysis than a denser carrier gas such as
nitrogen or argon. Additional benefits with the use of hydrogen as a carrier gas include

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the apparent ability of hydrogen to counteract trace amounts of oxygen (as opposed to
nitrogen and helium, which accumulate trace amounts of oxygen).

1.1.2 Gas Purifiers.

ac
m
Moisture and oxygen must be removed from the carrier gases by using appropriate
scrubbers. Even small amounts of oxygen or water can damage GC columns and
detectors. Thin films of stationary phase in capillary columns are especially vulnerable
ar
to oxidation or hydrolysis. This is good practice for any phase but is essential for very
polar phases. To ensure moisture-free gas (levels below 10 ppb), install a molecular
Ph

sieve 5A drying tube and an indicating tube, in series, in the line. A gas purifier
specifically designed to ensure maximum gas purity should be inserted in the carrier gas
line ahead of the injection port.
of

Oxygen free carrier gas is critically important in GC for both packed and capillary
columns. Problems encountered by oxygen entering the system are evident as
excessive column bleed caused by oxidation of the liquid phase, short column life, loss
ol

of sensitivity in electron-capture operation, and loss of column retention from phase

breakdown.
ho
Sc

Figure 3. Syringe

17
1.1.3 Sample introduction and the injection chamber
The sample, usually in the form of a solution in the order of 0.5 µl, is introduced into the
chromatograph with a micro syringe (Fig. 3).

Besides its role as an inlet for the sample, the injector has to vaporize and mix the sample
with the carrier gas before the sample enter the head of the column.

1.1.4 Oven
The gas chromatograph has oven with sufficient volume to hold the column easily and

0
which can be heated to desired temperature (between 40 and 450°C, stabilized to within

2
0.1°C).

20
1.1.5 Column
Separation of the sample components takes place in packed or open tubular columns
through which the carrier gas flows continuously. The separation column is placed

y
immediately after the injection port and any attendant sample splitter. The separation

ac
column contains the stationary phase, which can be either (1) an adsorbent (GSC) or (2)
a liquid distributed over the surface of small-diameter particles or the interior of capillary
tubing. Columns are made of metal (stainless steel, copper, or aluminum), glass, or fused
m
silica. Since inertness is of prime importance, glass and fused silica have become
increasingly popular; stainless steel is easier to handle and coil.
ar
GC columns can be divided into three broad categories: (1) packed columns, (2) open-
Ph

tubular columns, and (3) micro-packed columns. Two factors, selectivity and efficiency,
should be considered when comparing different types of columns.

1.1.5.1 Packed Columns.

of

Packed columns for analytical work are available in diameters of 2, 3, or 4 mm. If the
stationary phase is a liquid, it is held on the surface of an inert solid support. In GLC the
support should not take part in the separation. Especially with low liquid loadings (10%)
ol

and nonpolar phases, the support must be thoroughly inactivated when polar compounds
are to be analyzed. Adsorbents such as silica gel and molecular sieves are used for the
ho

separation of gases. The porous polymers, like the Porapaks, HayeSep, GasChrom,
Tenax-GC, and the Chromosorb Century Series, are used in special field applications such
as the analyses of solvents, acids, and alcohols in water. The analysis temperature is
Sc

often 50 to 100°C higher than that when a liquid phase is used. Carbopack and Graphpac
graphitized carbons are ideal for analyzing many kinds of C1 and C10 compounds,
including alcohols, free acids, amines, ketones, phenols, and aliphatic hydrocarbons.

The general performance characteristics of packed columns: Efficiency (plates per meter)
is a function of particle size and is about equal to that of a capillary column. Selectivity is
excellent due to the very wide range of phases that are available, including adsorbents
(GSC). Packed columns are applicable for all gases, are excellent for preparative-scale
analysis, but very limited for trace analysis. The practical length needs to be fairly short
due to low permeability—a major weakness. Speed of analysis is the slowest of all column
types. Compatibility with a mass spectrometer is very limited.
18
1.1.5.2 Stationary Phase (liquid type)
• Polysiloxanes
Polysiloxanes (also known as silicone oils or gums) are the most widely used for
capillary column because of their wide temperature range (-50 < T < 325°C).
Approximately 20 types of polysiloxane phases have been commercialised worldwide.

• Polyethylene glycols
The most widely known example of this family of compounds is Carbowax®. These
polar polymers can be used for deposition, impregnation or as bonded phase (60 < T

0
< 260°C, depending on column diameter and film thickness).

2
20
1.1.6 Stationary phases (solid type)
These phases are composed of adsorbing materials: molecular sieves, alumina, porous
glass and gels, and graphitized carbon black.

y
1.1.7 Detectors

ac
Some detectors are universal; that is, they are sensitive to almost every compound that
elutes from the column. However, most detectors are sensitive to a particular type a
compound. These are called selective detectors. Detector can be classified into two group
m
depending on whether they provide only retention time information or plus structural
information of an analyte.
ar
The ideal detector for gas chromatography has the following characteristics:
Ph

a. Adequate sensitivity.
b. Good stability and reproducibility.
c. A linear response to solutes that extends over several orders of magnitude.
of

d. A temperature ranges from room temperature to at least 400°C.

e. A short response time that is independent of flow rate.
f. High reliability and ease of use.
ol

g. Non-destructive of sample.
1.1.7.1 Thermal conductivity detector (TDC)
ho

This universal detector has been in use since the beginning of gas chromatography and
has been essential to the separation technique. Its basic operating principle relies on the
thermal conductivity of gaseous mixtures. In the steady state, a temperature equilibrium
Sc

exists, which depends on the resistance and which in turn is a function of the thermal
conductivity of the gas and of electrical current flowing through the filament. When a solute
elutes from the column there is a change in the composition of the mobile phase and thus
in the thermal conductivity. This result in a deviation from thermal equilibrium, causing a
variation in the resistance of one of filaments. This variation is proportional to the
concentration of analyte, provided its concentration in the mobile phase is low.

1.1.7.2 Flame ionisation detector (FID)

This detector, considered to be universal for the analysis of organic compound, appears
ideal for gas chromatography. The gas flow exiting the column passes through a small

19
burner fed by hydrogen and air. This detector essentially destroys the sample. Combustion
of the organic compounds flowing through the flame creates charged particles that are
responsible for generating a small current between two electrodes (voltage differential of
100-300 V). mFor organic compounds, the intensity of the signal is considered to be
proportional to the mass flow of carbon.

1.1.7.3 Nitrogen phosphorus detector (NPD)

This thermoionic is very sensitive to compounds that contain nitrogen or phosphorous.

1.1.7.4 Electron capture detector (ECD)

0
In this detector, a flow of nitrogen, ionised by electrons generated from a β-emitting
material of low energy (a few mCi of 63Ni), passes between two electrodes that are

2
maintained at a voltage differential of several hundred volts. At equilibrium, a base current

20
I0 is generated. If an organic substance M containing chlorine or fluorine passes through
the cell, it will capture some of the electrons leading to a decrease in the stationary current
and hence to a reduction in the signal. The ECD is well suited as a selective detector for

y
compounds with high electron affinity and limited linear response. ECD detector is mainly

ac
used for analysis of chlorine-containing pesticides.

1.1.7.5 Flame photometry detector (FPD)

m
The flame photometry detector is specific for compounds containing sulphur or
phosphorous.
ar
1.1.7.6 Detectors yielding structural information Atomic emission detector
Ph

It is possible to extend the principle of photometric emission (FPD) by replacing the flame
with a microwave plasma that has a temperature high enough to induce any element to
radiate light. This is equivalent to atomic emission where each solute is atomized and gives
rise to specific emission bands
of

1.1.7.7 Other detector

A mass spectrometric detector (MSD) can be placed at the end of the column thus
ol

producing fragmentation spectra of each of the eluting compounds.

ho

1.2 Analysis of less-volatile samples.

It has been said in the past that the use of GC was limited by the volatility of the solute.
This was not strictly accurate, since the limiting factor was more often the volatility of the
Sc

stationary phase. Bonded non-polar phase capillary column with upper limits of 325°C and
polar-phase column capable of working up to 230°C are available this moment, and a
number of manufacturers now offer variants of the non-polar or slightly polar capillary
column which are rated for use up to 480°C.

1.3 Derivatization.
Derivatization may be an excellent way of making a compound sufficiently volatile for GC
analysis. The objective of derivatization is to increase the volatility of the analyte.
Compounds which exhibit intramolecular H-bonding, e.g. alcohols, phenols, carboxylic
acids, and amines, usually have higher boiling points than might be anticipated on the

20
basis of their molecular weight. Clearly if the H-bonding could be eliminated then the
compound would become more volatile, making it more suitable for GC analysis. The most
common derivatization methods involve acetylation of alcohols, phenols, and amines using
acetic anhydride in the presence of an acid catalyst.

2. APPARATUS
Gas Chromatograph Hitachi G-5000A, Hitachi D-2500 Chromato-Integrator, Whatman
hydrogen generator, air compressor, OV-1 column, nitrogen gas, microsyringe 10 µl,
flowmeter, volumetric glassware.

0
3. CHEMICALS

2
Ethanol & propanol pro analysis.

20
4. PROCEDURE
4.1 Standards and Sample Preparation

y
a. Prepare the following standards in a 10-mL volumetric flask. Fill the flasks to the
mark with double distilled water after adding ethanol and mix well. (volume in

ac
mL): m
% ethanol (v/v) Vol. ethanol Vol. Distilled Water
1 0,1 0,9
ar
2 0,2 0,8
3 0,3 0,7
4 0,4 0,6
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5 0,5 0,5
6 0,6 0,4
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b. Pipette 5 mL of your sample into a 10-mL volumetric flask. Add 0.1 mL propanol
and fill to the mark with double distilled water. Mix well.
ol

4.2 GC Determination of Ethanol

a. Measure the standard and sample with the following GC system:
ho

- Column : Porapaq Q
- Detector : FID
Sc

- Carrier gas : Nitrogen

- Flow rate : 20 mL/min
- Temperature of detector : 225ºC
- Temperature of injector : 175ºC
- Temperature of column : 165ºC
b. Inject 2 L of pure ethanol into the GC system, obtain a chromatogram and record
the retention time of ethanol.
c. Inject 2 L of pure propanol into the GC system, obtain a chromatogram and
record the retention time of propanol.
d. Individually inject 2 L of each standard, obtain a chromatogram for each one and

21
 record the retention time and peak area of ethanol and propanol.
e. Inject 2 L of the sample into the GC system; repeat 3 times.
f. Before injected into the GC system, sample and standards must be filtered using
filter membrane 0.45 m.

4.3 Calculations
a. For each standard (1%, 2%, 3%, 4%, 5%, and 6% ethanol), calculate the ratio of
peak area of ethanol / peak area of propanol.
b. Make a calibration graph of the peak area ratio versus % ethanol. Fit a line, and
show the line and the equation on a calibration graph.

0
c. Calculate the peak area ratio (ethanol / propanol) for the sample.

2
d. Calculate the % ethanol of the sample using the equation obtained from the
calibration graph.

20
e. Finally, calculate the concentration of the original unknown solution you were
given. The solution tested was diluted (5 mL of original unknown diluted to a total
of 10 mL), so you need to calculate the original concentration before dilution.

y
f. Repeat the sample calculation for each injection you did, then calculate the

ac
average, standard deviation, and % coefficient of variation.
g. Repeat the calculation without use the propanol as an internal standard.
h. Compare the calculations.
m
ar
V. REFERENCES
Ph

Rouessac, F. and A. Rouessac, 2005, Chemical Analysis; Modern Instrumentation

Methods and Techniques, John Wiley & Sons, London, 23-37.
of

Shriner, R. L., C.K.F. Hermann, T.C. Morrill, D.Y. Curtin, and R.C. Fuson, 2004, The
systematic Identification of Organic Compounds, 8th ed., John Wiley & Sons,
Singapore, 90-99.
ol

Skoog, D.A, F.J. Holler, and T.A. Nieman, 1998, Principles of Instrumental Analysis,
5th ed., Harcourt College Publ., Orlando. 701-722.
ho

Watson, G., 2001, Pharmaceutical Analysis, Churchill Livingstone, London, 207-234.

Sc

22
 FLAME SPECTROSCOPY

1. INTRODUCTION
When a solution containing a suitable compound of the metal to be analyzed is aspirated
into a flame, the following events occur in rapid succession:
1. evaporation of solvent leaving a solid residue
2. vaporization of the solid with dissociation into its constituent atoms, which initially, will
be in the ground state; and
3. some of these gaseous metal atoms may be excited by the thermal energy of the flame

0
to higher energy levels, which is sufficiently high to permit the emission of radiation
characteristic of the metal

2
20
This is the basis of flame emission spectroscopy (FES) which was formerly referred to as
flame photometry. The resulting emission spectrum thus consists of lines originating from
excited atoms or ions.

y
Ground-state atoms are capable of absorbing radiant energy of their own specific resonance

ac
wavelength, which in general is the wavelength of the radiation that the atoms would emit if
excited from the ground state. Hence if light of the resonance wavelength is passed through
m
a flame containing the atoms in question, then part of the light will be absorbed, and the
extent of absorption will be proportional to the number of ground-state atoms present in the
ar
flame. This is the underlying principle of atomic absorption spectroscopy (AAS). Atomic
fluorescence spectroscopy (AFS) is based on the re-emission of absorbed energy by free
atoms.
Ph

1.1 Sample Atomization techniques

1.1.1 Flame atomization
of

In a flame atomizer, a solution of the sample is nebulized by a flow of gaseous oxidant,

mixed with a gaseous fuel, and carried into a flame where atomization occurs (Figure 1).
ol

1.1.2 Electrothermal atomization

In electrothermal atomizers, a few microliters of sample are first evaporated at a low
ho

temperature and then ashed at a somewhat higher temperature in an electrically heated

graphite tube. After ashing the current is rapidly increased to several hundred amperes,
which causes the temperature to soar to perhaps 2000 ºC to 3000 ºC; atomization of the
Sc

sample occurs in a period of a few millisecond to seconds.

23
 2 0
y 20
ac
m
Figure 1. Processes occurring during atomization
ar
Ph
of
ol
ho
Sc

Figure 2. Graphite furnace

24
1.1.3 Specialized atomization techniques
- Glow discharge atomization
- Hydride atomization
- Cold-Vapor atomization

1.2 Radiation sources

1.2.1 Hollow Cathode lamps
The most common source for atomic absorption measurements is the hollow cathode
lamp, such as the one shown in Figure 3. This type of lamp consists of a tungsten anode
and a cylindrical cathode sealed in a glass tube that is filled with neon or argon at a

0
pressure of 1 to 5 torr. The cathode is constructed of the metal whose spectrum is desired

2
or serves to support a layer of that metal.

y 20
ac
m
ar
Figure 3. Schematic of a Hollow Cathode lamp

1.2.2 Electrodeless Discharge lamps

Ph

Electrodeless Discharge lamps (EDLs) are useful sources of atomic line spectra and
provide radiant intensities that are usually one to two orders of magnitude greater than
hollow cathode lamps. A typical lamp is constructed from a sealed quartz tube containing
of

a few torr of an inert gas such as argon and a small quantity of the metal (or its salt) whose
spectrum is of interest. The lamp contains no electrode but instead is energized by an
intense field of radio-frequency or microwave radiation.
ol
ho
Sc

Figure 4. Electrodeless Discharge lamp

1.3 Spectrophotometers
Photometric emission measurements are carried out using either atomic absorption
25
spectrophotometers without the light source, or flame photometers. The latter are simple
instruments whose price is ten times less that of atomic absorption spectrophotometers.
Flame photometers are designed to make measurements on five or six elements. They
include interchangeable colored filters that can isolate a spectral band, including the
chosen emission line. Flame photometers are widely used for certain applications in quality
control such as measurements of alkaline and alkaline earth metals in substances. Some
improved models’ possess two measuring cells that allow a direct reading of emission
intensity ratios by comparison to an internal standard, allowing concentration
determination. Response linearity is limited to concentration between 10 and 100 ppm, so
large dilutions of samples are typically required.

2 0
y 20
ac
m
ar
Ph
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Figure 5. Typical atomic absorption spectrophotometers: (a) single-beam design and

ho

(b) double- beam design

2. APPARATUS
Sc

SPECTRAA 50/55 Screen AAS, volumetric glassware, washing bottle.

3. CHEMICALS
Cu stock solution, deionized water, nitrous oxide gas, acetylene.

4. PROCEDURE
Standards and Sample Preparation
a. Prepare six volumetric flasks.
b. Pipette 0.5, 1, 1.5, 2, 2.5, and 3 mL of 100 ppm Cu stock solution and 1 mL of

26
 sample into the volumetric flask. Dilute to 100 mL with deionized water.

Determination of Copper (Cu)

a. Aspirate each of your 6 Cu standard solutions and record their absorbance values.
b. Prepare a calibration curve by plotting the absorbance value of standards as a
function of Cu concentration.
c. Aspirate deionized water in between each Cu solution to clean the unit out.
d. Determine the concentration of Cu element in your sample from the graph.

5. REFERENCES

0
Rouessac, F., and A. Rouessac, 2005, Chemical Analysis; Modern Instrumentation

2
Methods and Techniques, John Wiley & Sons, London.

20
Skoog, D.A, F.J. Holler, and T.A. Nieman, 1998, Principles of Instrumental Analysis,
5th ed., Harcourt College Publ., Orlando.

y
ac
m
ar
Ph
of
ol
ho
Sc

27
 SPECTROFLUOROMETRY

1. INTRODUCTION
Photoluminescence is a type of optical spectroscopy in which a molecule is promoted to
an electronically excited state by absorption of ultraviolet, visible, or near infrared
radiation. The excited molecule then decays back to the ground state, or to a lower-lying
excited electronic state, by emission of light. The emitted light is detected.
Photoluminescence processes are subdivided into fluorescence and phosphorescence.

0
1.1 Information Available from Fluorescence Measurements

2
Two basic types of spectra can be produced by a fluorescence spectrometer. In a
fluorescence spectrum, or emission spectrum, the wavelength of the exciting radiation is

20
held constant (at a wavelength at which the analyte absorbs) and the spectral distribution
of the emitted radiation is measured. In an excitation spectrum, the fluorescence signal, at
a fixed emission wavelength, is measured as the wavelength of the exciting radiation is

y
varied. Because an analyte can fluoresce only after it has absorbed radiation, an excitation

ac
spectrum identifies the wavelengths of light that the analyte is able to absorb. Thus, subject
to certain constraints, the excitation spectrum of a molecule should be the same as its
UV/Vis absorption spectrum.
m
The excitation spectrum for a compound should not change as the emission wavelength
ar
is varied. Whenever the excitation spectrum varies with choice of emission wavelength,
there is good reason to believe that two or more different substances are responsible for
Ph

the observed fluorescence. The maximum in the fluorescence spectrum of a compound

occurs at longer wavelength than the maximum in the absorption spectrum. The
wavelength difference between the absorption and fluorescence maxima is called the
Stokes shift. Often, the Stokes shift is large (20 to 50 nm), especially for polar solutes in
of

polar solvents. There is often some overlap, but not a great deal, between the absorption
and fluorescence spectra of a compound. Both spectra may exhibit wavelength shifts
whenever the solvent is changed; again, these effects are largest for polar solutes
ol

dissolved in polar, hydrogen-bonding solvents.

ho

Fluorescence, which often occurs in cyclic, rigid molecules that contain electrons, is
enhanced by the presence of electron-donating groups and decreased by electron-
withdrawing groups (Fig. 2.). It also depends on pH and solvent. Non-rigid molecules, on
Sc

the other hand, easily lose all of their absorbed energy by degradation and vibrational
relaxation.

28
 2 0
20
Figure 1. Representation on overlaid graphs of the absorption and fluorescence spectra of
a triene.

y
ac
m
ar
Ph
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Figure 2. Fluorescent aromatic compounds. The compound names are followed by their
fluorescent quantum yields, whose values are obtained by comparison to compounds of
ol

known fluorescence.
ho

1.2 Interferences
It is useful to consider interferences in two classes: additive and multiplicative. In additive
interference, background fluorescence is emitted by concomitants in the sample or
Sc

contaminants in solvents or glassware, causing a finite blank. In multiplicative interference,

the interferent does not itself fluoresce, but causes the fluorescence signal for the analyte
to be smaller or (occasionally) larger than that observed in the absence of the interferent.
One important phenomenon—quenching—can simultaneously cause additive and
multiplicative interference. It is generally easier to correct for multiplicative interference (by
standard additions, for example) than additive interference.

1.3 Instrumentation
In fluorescence, the sample behaves like a light source, emitting in all directions. The
emitted light is usually monitored perpendicular to the primary excitation source. For
strongly absorbing solutions, measurements can be made on-axis with the incident
29
radiation. For opaque or semi- opaque samples, a frontal measurement at various angles
is recommended (Fig. 3). Commercial instruments use a xenon arc lamp with power of 150
to 800 watts as an excitation source. Measurement of the light intensity is carried out using
a photomultiplier tube or a photodiode. Two categories of fluorescence instruments are
available from manufacturers.

2 0
y 20
Figure 3. The geometry of a fluorimeter and layout of the different instruments

1.4 Fluorescence ratio fluorimeters (quantitation)

ac
m
Light emitted from the source converges on the excitation monochromator, which allows a
narrow bend of wavelengths to be selected (15 nm) to induce fluorescence of the sample
ar
solution in the measurement cell. The emitted light, which is observed perpendicular to the
direction of the incident beam, passes through the emission monochromator, allowing the
selection of a narrow bend of wavelengths for measurement (Fig. 4). The simplest
Ph

instruments have a double compartment for measurement. This allows the sample solution
and a standard fluorescent reference solution to be put into the optical path.
of
ol
ho
Sc

Figure 4. Optical scheme of a spectrofluorometer have two detectors, one of which is used
to control the intensity of the light source. A fraction of the incident beam is reflected by
the beam splitter and monitored by a photodiode to control the intensity of the incident
beam. Comparison of the signals obtained from both detectors allows the elimination of
any drift in the light source. This procedure, for single beam instruments, gives
approximately the same stability as with double beam instruments.

30
 Used as standards method are generally Quinine sulphate, rhodamine B or 2-
aminopyridine solutions. In order to eliminate fluctuations due to the radiation source and
other instruments parameters, comparative measurements are used. Instrument
sensitivity is commonly expressed in terms of the signal to noise ratio. The nature of the
solvent, temperature, the pH and the concentration can affect the intensity of fluorescence.

1.5 Spectrofluorometers.
In contrast to the preceding set-up, spectrofluorometers record an entire fluorescence
spectrum. Each of the two motorized monochromators can scan a spectral band. It is
possible to record the emission spectrum while maintaining a constant excitation
wavelength or to record the excitation spectrum while maintaining a constant emission

0
wavelength.

2
20
2. APPARATUS
Spectrofluorometer Shimadzu RF-540, fluorometric cuvette, volumetric glassware,
analytical balance, beaker glass, dropper pipette, spatula, wash bottle, lens paper.

y
3. CHEMICALS
Sulfuric acid, quinine sulfate, distilled water.

ac
m
4. PROCEDURE
Determination of Quinine
a. Prepare 1000 mL of 0.1 N H2SO4.
ar
b. Prepare a quinine stock solution by weighing accurately about 10 mg of quinine sulfate,
and transferring it into a 100-mL volumetric flask and diluting to the mark with 0.1 N
Ph

H2SO4 (concentration of stock solution will be 100 ppm). From this first stock solution,
prepare the second quinine stock solution of 1 g/mL (1 ppm).
c. Prepare five standard quinine solutions of 0.1, 0.15, 0.2, 0.25, 0.3 ppm by an
of

appropriate dilution of the second quinine stock solution with 0.1 N H2SO4.
d. Pour about 10 mL of sample to a clean and dry beaker and shake it. Then pipette 5.00
mL sample to 100-mL volumetric flasks and make it up to the mark with 0.1 N H2SO4.
ol

e. Determine the excitation wavelength of quinine.

f. Determine the emission wavelength of quinine.
ho

g. Measure the fluorescence intensity of the standard and sample solutions

h. Develop a calibration curve (the concentration of standard [x-axis] vs fluorescence
Sc

intensity [y-axis]) and report the concentration of quinine in the sample in ppm.

5. REFERENCE
Rouessac, F. and A. Rouessac, 2005, Chemical Analysis; Modern Instrumentation
Methods and Techniques, John Wiley & Sons, London, 221-233.

Skoog, D.A, F.J. Holler, and T.A. Nieman, 1998, Principles of Instrumental Analysis, 5th
ed., Harcourt College Publ., Orlando, 300-351.

Watson, G., 2001, Pharmaceutical Analysis, Churchill Livingstone, London, 133-142

31
 POTENTIOMETRY

1. INTRODUCTION
Potentiometric methods of analysis are based on measuring the potential of
electrochemical cells without drawing appreciable current. An electrochemical cell consists
of two conductors called electrodes, each of which is immersed in an electrolyte solution.
The two electrodes are different and must be separated to avoid direct reaction between
the reactants. Each of these electrodes constitutes a half-cell which the absolute values
for individual half-cell potentials cannot be determined in the laboratory, only relative cell
potentials can be measured experimentally.

2 0
y 20
ac
m
ar
1. The reference electrode in is a half-cell with an accurately known electrode potential
Ph

(Eref) that is independent of the concentration of the analyte or any other ions in the
solution under study.
2. The indicator electrode, which is immersed in a solution of the analyte, develops a
potential (Eind) that depends on the activity of the analyte. There are 3 kind of indicator
of

electode : metallic, membrane and ion selective field transistors. Most indicator
electrodes used in potentiometry are selective in their responses.
3. Junction Potential (Ej) is a voltage difference developed at the interface of two
ol

dissimilar electrolyte solutions are placed in contact. This small, unknown voltage
(usually a few millivolts) exists at each end of a salt bridge connecting two half cells.
ho

The junction potential puts a fundamental limitation on the accuracy of direct

potentiometric measurements (because we usually do not know the contribution of the
junction to the measured voltage)
Sc

4. Salt bridge used to prevents the components of the analyte solution from mixing with
those of the reference electrode. A potential develops across the liquid junctions at
each end of the salt bridge. These two potentials tend to cancel one another if the
mobilities of the cation and the anion in the bridge solution are approximately the
same. Potassium chloride is a nearly ideal electrolyte for the salt bridge because the
mobilities of the K+ ion and and the Cl- ion are nearly equal. The net potential across
the salt bridge, Ej, is thereby reduced to a few millivolts or less. For most
electroanalytical methods, the junction potential is small enough to be neglected.

32
One of most applicable usage of potentiometric is determining ion in solution (p-ion such
as pH, pCa, pNO3, etc). The basic principle of this measurement is to measure the
potential that appears across a thin glass membrane that separates two solutions with
different hydrogen ion concentrations. In this experiment we will use potentiometer that is
used for measure the concentration of hydrogen ion in solution (called pH meter).

1.1 pH Meter
The first potentiometric cell consists of a glass indicator electrode (membrane indicator
electrode) and a reference electrode (calomel electrode) immersed in the solution of
unknown pH. The indicator electrode consists of a thin pH-sensitive glass membrane

0
sealed onto one end of a heavy-walled glass or plastic tube. A small volume of dilute
hydrochloric acid saturated with silver chloride is contained in the tube. The inner solution

2
in some electrodes is a buffer containing chloride ion. A silver wire in this solution forms a

20
silver/silver chloride reference electrode, which is connected to one of the terminals of a
potential- measuring device. The calomel electrode is connected to the other terminal.

y
ac
m
ar
Ph
of

Figure 1. Schematic of the previous Potentiometer.

ol

And the schematic of the potential is :

ho
Sc

Figure 2. Potential developed when pH is being determined with glass electrode.

33
In the glass electrode (indicator electrode), the concentration (and the activity) of protons
inside the membrane is constant. The concentration outside the membrane is determined
by the activity of hydrogen ions in the analyte solution. This concentration difference
produces the potential difference measured with a pH meter. The potential of glass
electrode (Eind) will be :
𝐸𝑖𝑛𝑑 = 𝐸𝑏 + 𝐸𝐴𝑔/𝐴𝑔𝐶𝑙 + 𝐸𝐴𝑠𝑦
Easy is the asymmetry potential that is developed when two identical solution (the same
pH of solution) is in contact with membrane. The asymmetry potential can be corrected
with calibrating electrode in one or more solution of known pH.

0
The internal and external reference electrodes are just the means of making electrical
contact with the two sides of the glass membrane and that their potentials are essentially

2
constant except for the junction potential, which depends to a small extent on the

20
composition of the analyte solution. The potentials of the two reference electrodes depend
on the electrochemical characteristics of their respective redox couples, but the potential
across the glass membrane depends on the physicochemical characteristics of the glass

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and its response to ionic concentrations on both sides of the membrane.

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There are four potential that is produced when a pH is being determined (Figure 7.2). The
potentials of EAg|AgCl and ESCE, (both are references electrode) are constant. The
junction potential, Ej, across the salt bridge that separates the calomel electrode from the
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analyte solution are found in all cells. The boundary potential (Eb) will vary with the pH of
the analyte solution. The two reference electrodes provide electrical contacts with the
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solutions so the changes in the boundary potential can be measured.
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The boundary potential is produced between the differences two surfaces of the glass
membranes when its surface in contact with solution (E1 and E2; Figure 7.3 below). The
hydrogen ion concentrations in the solutions on the two sides of the membrane control the
positions of the equilibria which is related to the activities of hydrogen ions in each of the
of

solutions by the Nernst-like equation:

𝑎
Eb = E1 – E2 = 0.05916 log 𝑎1
2
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Whether the hydrogen ion activity of the internal solution(a2) is held constant so the
equation will be
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𝐸𝑏 = 0.0591 log 𝑎1 − 0.0591 6log 𝑎2

𝐸𝑏 = 𝐶𝑜𝑛𝑠𝑡𝑎𝑛𝑡𝑎 + 0.0591 6log 𝑎1
𝐸𝑏 = 𝐶𝑜𝑛𝑠𝑡𝑎𝑛𝑡𝑎 − 0.05916𝑝𝐻
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When identical solutions and reference electrodes are placed on the two sides of a glass
membrane, the boundary potential should in principle be zero. But in real situation, a small
asymmetry potential developed gradually with time.

The Potential Indicator then:

Eind = Eb + EAg/AgCl + EAsy
Eind = Constanta – 0.05916 pH + EAg/AgCl + EAsy
Eind = Constanta2 – 0.05916 pH

34
And
Eobserved (V) = Ecell = Eind – Eref + Ej
Ecell = (Constanta2 – 0.05916 pH) – Eref + Ej
𝐸𝑐𝑒𝑙𝑙−(𝐸𝑖−𝐸𝑟𝑒𝑓+𝐶𝑜𝑛𝑠𝑡𝑎𝑛𝑡𝑎2)
pH = − 0.05916
𝐸𝑐𝑒𝑙𝑙−(𝐶𝑜𝑛𝑠𝑡𝑎𝑛𝑡𝑎3)
pH =− 0.05916

The most common configuration for measuring pH with a glass electrode nowadays is a
combination electrode. In this arrangement, the glass electrode and its Ag/AgCl internal
reference electrode are positioned in the center of a cylindrical probe. Surrounding the glass

0
electrode is the external reference electrode, which is most often of the Ag/AgCl type

2
(Figure 3).

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Figure 3. Combination Electrode and Schematic Electrochemical cells

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1.2 Potentiometric titration

The method can be applied to any titrimetric reaction for which an indicator
electrode is available to follow the activity of at least one of the substances involved.
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In a potentiometric titration, we measure the potential of a suitable indicator

electrode as a function of titrant volume. Titration results depend most heavily on
having a titrant of accurately known concentration. The potentiometric instrument
merely signals the end point and thus behaves in an identical fashion to a chemical
indicator. Another advantage of a titration is that the result is analyte concentration
even though the electrode responds to activity. The operator measures and records
the cell potential (in units of millivolts or pH) after each addition of reagent.

1.3 Location of the Equivalence Point

The critical problem in a titration is the recognition of the point at which the quantities of

35
reacting species are present in equivalent amounts – the equivalence point. The titration
curve can be followed point by point, plotting as the ordinate successive values of the
potential cell or pH versus the corresponding volume of titrant added as the abscissa.
Additions of titrant should be the smallest accurately measurable increments that provide
an adequate density of points, particularly in the vicinity of the equivalence point.

2 0
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Figure 4. Potentiometric Titration and the Titration curve of monoprotic acid with strong base
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Remember in Basic Pharmaceutical Analysis Lecture, that before the equivalence point,
there will be buffer region, in which Henderson-Hasselbalch equation will take place. And
at the point where the volume = ½ equivalence volume, the [A-] will be equal to [HA] and according
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to the equation above, pH = pKa. For triprotic acid, there will be three pKa values and the
calculation will be summarized in the figure below
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36
 The end point can be more precisely located from the first or second derivative curves.

0
Figure 5. Potentiometric titration curves: (a) experimental titration curve, (b) first

2
derivative curve, and (c) second derivative curve.

20
2. APPARATUS
pH METER BECKMAN 50, Burette, magnetic stirring bar, beaker glass, volumetric

y
glassware, wash bottle.

3. CHEMICALS

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Sodium Hydroxide (0.1 M), phosphoric acid (0.05M), distilled water.
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4. PROCEDURE
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4.1 Calibrating pH Meter
a. Turn on the pH meter,
b. Rinse the electrode with distilled water, gently blot it dry with a tissue. Do not
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wipe it
c. because this action might produce a static charge on the glass
d. Dip the electrode in a first standard buffer and allow the electrode to equilibrate.
e. Press “STD” key in pH metter. Allow pH meter to measure pH automatically.
of

f. Wash the electrode with water, blot it dry, and immerse it in a second standard
buffer.
g. Press “STD” key in pH metter. Allow pH meter to measure pH automatically.
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h. Do calibration with minimum 3 standard buffer and press END or SAVE to finish
the calibration. Note the OFFSET and SLOPE of the electrode.
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4.2 Titration:
a. Pipette 10 mL of 0.05 M H3PO4 into a 100 mL beaker, add approximately 20
mL of distilled water.
b. Into the beaker, place magnetic stirring bar.
c. Immerse the electrode in solution.
37
 d. Keep the electrode high enough so that the stirring bar does not strike it.
e. Clean, rinse, and fill a 25-mL burette with 0.1 M NaOH (remembering to be
sure to rinse the burette with a little NaOH first before filling).
f. After all of the apparatus have been set, press the key. pH

g. After stops flashing, display will show pH of solution.

h. Record the initial pH of the sample

i. Start to titrate potentiometrically the solution with 0.1 M NaOH
j. With continuous stirring, small increments of 0.1 M NaOH are added from a burette.
k. The pH of the solution is measured

0
l. Record the pH and volume in the notebook after each portion has been added.

2
m. As the equivalence point of the titration is approached, pH changes rapidly,

20
so smaller increments of NaOH solution should be added.
n. After the titration finish, rinse the electrode with distilled water, and blot excess
o. Close the electrode with saturated KCl Solution

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4.3 Calculation

ac
a. Calculate the molarity of H3PO4.
b. Plot pH (y-axis) vs the volume of NaOH (x-axis).
m
c. Determine the first and second End Point Titration. The End Point Titration
can be determined manually:
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Ph
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Or using spreadsheet to generate the first and second derivative of titration

curve

d. Plot the first derivative curves. To make this curve, calculate the change
in pH (ΔpH) and the change in volume added (ΔV) for each pair of
consecutive additions. Calculate the first derivative (ΔpH/ΔV) for each pair
of points. Plot the first derivative (ΔpH/ΔV) on the y-axis vs the average
volume added on the x-axis. Determine the endpoint of the titration from
the maximum of the curve. The maximum in ΔpH/ΔV indicates the location
of the inflection point of the titration.

38
 2 0
ΔV = Vn – Vn-1

20
ΔpH = pHn + pHn-1
𝑉𝑛+𝑉𝑛−1
V (avge) = 2

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Ph
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e. Plot the second derivative curves. To make this curve, first calculate the change in
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the change of pH (Δ2pH) from consecutive values of a pH and calculate the change
in the change of volume (Δ2V) and plot. The second derivative passes through zero
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at the inflection point. Linear interpolation can be used to calculate the point at
which the second derivative is zero.
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39
 ΔV = V(avge)n – V(avge)n-1
Δ(ΔpH) = (ΔpH/ΔV)n − (ΔpH/ΔV)n-1
𝑉𝑛+𝑉𝑛−1
V (avge) = 2

2 0
20
f. Determine the pKa1, pKa2, and (if possible) pKa3 of H3PO4.

y
ac
5. REFERENCES
Rouessac, F, and A Rouessac, 2005, Chemical Analysis; Modern Instrumentation
Methods and Techniques, John Wiley & Sons, London
m
Skoog, D., West D., Holler FJ, Crouch SR, 2013, Fundamental of Analytical Chemistry
9th Ed, Brooks & Cole, New York.
ar
Billo, EJ, 2011, Excel for Chemist 3rd Ed, John Wiley & Sons, Canada
Harris, DC, 2010, Quantitative Chemical Analysis 8th Ed, W.H. Freeman and
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Company, New York.

http://www.chemtopics.com/aplab/diprotic.pdf
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40
 BIAMPEROMETRY

1. INTRODUCTION
From the previous module, we have learnt the basic principle and little application
potentiometry—in which voltage was measured in the absence of significant current. There
are another technique in electrochemistry where current play important role: electrolysis.

Electrolysis is a chemical reaction in which a voltage is applied to drive a redox reaction

that would not otherwise occur or in another words, the process in which a chemical
reaction is forced to occur at an electrode by an imposed voltage. One kind of electrolysis

0
is voltammetry. Voltammetry is a collection of techniques in which the relation between
current and voltage is observed during electrochemical processes (information about the

2
analyte is acquired by measuring current in an electrochemical cell as a function of applied

20
potential). When current proportional to analyte concentration is monitored at a fixed
potential, the technique is called amperometry.

y
There are several term in the voltammetry:
1. Polarization is the deviation of the electrode potential from the value predicted by the

ac
Nerst equation on the passage of current that is given by an overvoltage
(overpotential) which is potential greater than the theoretical value is required to give
m
a current of the expected magnitude
2. A depolarizer is a species that is easily reduced (or oxidized) and therefore “alters”
the polarization of the electrode. It helps maintain the potential of the working
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electrode at a relatively small constant value and prevents reactions that would occur
under more reducing or oxidizing conditions.
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3. An electroactive species is one that can be oxidized or reduced at an electrode.

4. A working electrode is the electrode at which the analytical reaction occurs
(oxidation or reduction occurs). The working electrode is equivalent to the indicator
electrode of two-electrode cells. Metal electrodes are polarizable electrode because
of

the potentials are easily changed when small current is flowing.

5. A reference electrode is an electrode that provides fixed reference potential with
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negligible current flow. A reference electrode (such as calomel or Ag|AgCl) is

nonpolarizable electrode, because its potential does not vary much unless a
significant current is flowing.
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Potentiostat is a device that control the voltage between the working and reference
electrodes.
Sc

Figure 1. Schematic of Amperometry Apparatus

41
The electrochemical cells that have been discussed are based on two electrodes: an
indicator electrode and a reference electrode. Current is measured between the two
electrodes. A cell with three electrodes is required for fine control of the electrochemistry.
The cells above have three electrode: reference electrode, working electrode, and an
auxiliary electrode (counter electrode). Auxiliary electrode that is the current-carrying
partner of the working electrode. Current flows between the working and auxiliary
electrodes. Voltage is measured between the working and reference electrodes. Usually,
in a two-electrode cell, the potential of the working electrode can drift as the reaction
proceeds. As it drifts, reactions other than the intended analytical reaction can take place.
In this case, the potentiostat maintains the working electrode at the desired potential, while
the auxiliary electrode potential drifts out of our control.

2 0
Voltammetry can be used to estimate the equivalence point of titrations if at least one of

20
the participants or products of the reaction involved is oxidized or reduced at a working
electrode. This method is called amperometric titration. In this case, the current at some
fixed potential in the limiting current region is measured as a function of the reagent
volume.

y
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Depending on the potential of the electrode and the voltammetric characteristics of the
chemical substances involved, the current may be proportional to the concentration of the
substances being titrated, to the concentration of the excess of reagent, or to the
m
concentration of one of the products of the reaction, or it may depend on two of these
concentrations. The titration curve is a plot of the limiting current against the volume of the
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reagent added. The end point is established by extrapolation to the intersection of the
lines. Figure 2 (a) represents a titration in which the analyte reacts at the working electrode
Ph

while the reagent does not. 2 (b) is typical of a titration in which the reagent reacts at the
working electrode and the analyte does not. 2 (c) corresponds to a titration in which both
the analyte and the titrant react at the working electrode.
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Figure 2. Typical amperometric titration curves. (a) Analyte is reduced; reagent

is not. (b) Reagent is reduced; analyte is not. (c) Both reagent and
analyte are reduced

There are two types of amperometric electrode systems: one uses a single polarizable
electrode coupled to a reference, while the other uses a pair of identical solid-state
polarizable electrodes immersed in a stirred solution. One of example of
amperometric with a pair of identical solid-state polarizable electrodes is
biamperometry.
42
Biamperometry is an application of voltammetry that can be used to estimate the
equivalence point of titrations, provided at least one of the participants or products of the
reaction involved is oxidized or reduced at the electrode. Biamperometric titration uses a
pair of identical solid-state microelectrodes (platinum-platinum electrodes) immersed in a
stirred solution. One of these electrodes functions as an anode and the other as a cathode.
The electrode system consists of a pair of twin platinum microelectrodes between which is
imposed a potential difference (0 – 200 mV). There is essentially no current, if no easily
reduced or oxidized species is present in the solution and the electrodes is completely
polarized. When easily reduced or oxidized species is present in the solution, one of the
electrodes is not polarized because the reduction or oxidation occurs in the presence of
this species (depolarizer). A current develops as a result of this reaction. The magnitude

0
of the current is, as in other amperometric methods, directly proportional to the

2
concentration of the excess reagent, so this method can be used to estimate the

20
equivalence point of titrations.

2. APPARATUS
Biamperometer Batrana, burette, magnetic stirring bar, beaker glass, volumetric

y
glassware, wash bottle.

3. CHEMICALS
ac
Sulfanilamide, ascorbic acid, sodium nitrite, hydrochloric acid, iodine, potassium iodide,
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sulfuric acid, arsenic trioxide.
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4. PROCEDURE
4.1 Titration of Sulfanilamide
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4.1.1 Prepare the following solutions:

a. Sodium nitrite (0.1 M): dissolve 7.5 g of sodium nitrite in water to make 1000
mL, and standardize the solution.
b. Sample solution: in a 500mL-beaker glass, about 400 mg of sulfanilamide
of

sample (accurately weighed, previously dried at 105°C for 3 hours) is dissolved

by stirring in 20 mL of hydrochloric acid and 50 mL of water, and cool to 15°C.
4.1.2 Analysis
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a. Maintain the temperature of the sample solution at about 15°C.

b. Into the beaker glass containing sample, plunge a magnetic stirring bar and then
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immerse the electrode with its tip to be high enough so that the stirring bar does
not strike it.
c. Fill a 25-mL burette with 0.1 M sodium nitrite (assure to rinse the burette with a
Sc

little of sodium nitrite first before filling).

d. Set up the burette such that its tip below the surface of the solution to avoid
atmospheric oxidation of the sodium nitrite.
e. Set the potential of the electrode in the range of 100-110 mV.
f. Start to titrate the sample solution, first by addition of about one half of the total
of the titrant (0.1 M sodium nitrite) needed, and record the current (mA) and the
volume of the titrant added.
g. While stirring, small increments of 0.1 M sodium nitrite are added from the
burette. After each addition of sodium nitrite, record the current (mA) and the
volume of the titrant added. When the end point of the titration is within about 1
43
 mL of titrant, add the titrant in 0.1 mL portions, and allow 1 minute between
additions.
h. Plot Current (y-axis) vs the volume of sodium nitrite (x-axis). Determine the
equivalent point of the titration.
i. Calculate the content of sulfanilamide in the sample (% b/b).

4.2 Determination of Ascorbic Acid Content of a Sample

4.2.1 Prepare the following solutions:
a. Solution of 2 N sulfuric acid
b. Solution of 0.1N iodine: dissolve about 14 g of iodine in a solution of 36 g of
potassium iodide in 100 mL of water, add 3 drops of hydrochloric acid, dilute

0
with water to 1000 mL, and standardize the solution. Store up in an amber

2
colored, glass-stoppered bottle.

20
c. Sample solution: accurately weigh about 200 mg of ascorbic acid sample, and
transfer to a suitable beaker. Add 100 mL of distilled water and 25 mL of 2 N
sulfuric acid, stir until dissolved.

y
4.2.2 Analysis

ac
a. Into the beaker (sample solution), place magnetic stirring bar.
b. Immerse the electrode in solution. Keep the electrode high enough so that the
stirring bar does not strike it.
m
c. Clean, rinse, and fill a 25-mL burette with 0.1 N iodine (remembering to be
sure to rinse the burette with a little of iodine first before filling).
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d. Set the potential at the electrode about 50 mV.
e. Start to titrate the sample solution with 0.1 N iodine.
f. With continuous stirring, small increments of 0.1 N iodine are added from a
Ph

burette.
g. Record the current (mA) and volume of the titrant in the notebook after each
portion of iodine has been added.
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h. When the titration is within 1 mL of the end point, add the titrant in 0.1 mL
portions, and allow 1 minute between additions.
i. Plot Current (y-axis) vs the volume of iodine (x-axis). Determine the equivalent
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point of the titration.

j. Calculate the ascorbic acid content of the sample (% b/b).
ho

5. REFERENCES
Skoog, D.A, F.J. Holler, and T.A. Nieman, 1998, Principles of Instrumental Analysis, 5th
ed., Harcourt College Publ., Orlando, 639-670.
Sc

Rouessac, F, and A Rouessac, 2005, Chemical Analysis; Modern Instrumentation

Methods and Techniques, John Wiley & Sons, London

Skoog, D., West D., Holler FJ, Crouch SR, 2013, Fundamental of Analytical Chemistry
9th Ed, Brooks & Cole, New York.

Harris, DC, 2010, Quantitative Chemical Analysis 8th Ed, W.H. Freeman and Company,
New York.

Zutshi, K, 2006, Introduction to Polarography and Allied Techniques, New Ages

International Ltd, New Delhi
44