Quantitative Analytical Chemistry

Instrumental Analysis

Atomic Absorption Spectrometric Analysis of Fluoroquinolones

Supervised by

Prof.Dr/ Hesham Salem

Prepared by

Shrouq Ashraf Elsayed

2020/2021
 List of Contents
Introduction ...........................................................................1

Instrumental chemistry .............................................................3

Review ..................................................................................4

Atomic Absorption Spectrometric ................................................4

Atomic Absorption Spectrometric Analysis of Fluoroquinolones ........7

Ciprofloxacin, Amoxicillin and Diclofenac Sodium .........................7

Conclusion .......................................................................... 12

References ........................................................................... 13
 Table of Figures
Figure 1 Molecular structures of ciprofloxacin (a), amoxycillin (b) and diclofenac
sodium (c). ........................................................................................... 7

Figure 2 Effect of temperature on the oxidation reactions of the drugs, ciprofloxacin 0.4
µg ml-1, amoxycillin 0.5 µg ml-1, and diclofenac sodium 0.3 µg ml-1. ................ 10

Figure 3 Effect of heating time on the oxidation reactions of the drugs, ciprofloxacin
0.4 µg ml-1, amoxycillin 0.5 µg ml-1, and diclofenac sodium 0.3 µg ml-1. ........... 10

Figure 4 Effect of pH on the oxidation reactions of the drugs, ciprofloxacin 0.4 µg ml-
1, amoxycillin 0.5 µg ml-1, and diclofenac sodium 0.3 µg ml-1. ........................ 11

Figure 5 Effect of iron(III) concentration on the drugs, ciprofloxacin 0.4 µg ml-1,

amoxycillin 0.5 µg ml-1, and diclofenac sodium 0.3 µg ml-1............................. 11

Table of Tables
Table 1 Assay of commercial tablet formulations by the AAS method and the official
method ................................................................................................ 9

Table 2 Intraday and interday assay of ciprofloxacin, amoxycillin and diclofenac

sodium ................................................................................................ 9
Introduction
Analytical chemistry has been important since the early days of chemistry,
providing methods for determining which elements and chemicals are
present in the object in question. During this period significant
contributions to analytical chemistry include the development of
systematic elemental analysis by Justus von Liebig and systematized
organic analysis based on the specific reactions of functional groups.
The first instrumental analysis was flame emissive spectrometry developed
by Robert Bunsen and Gustav Kirchhoff who discovered rubidium (Rb)
and caesium (Cs) in 1860
Most of the major developments in analytical chemistry take place after
1900. During this period instrumental analysis becomes progressively
dominant in the field. In particular many of the basic spectroscopic and
spectrometric techniques were discovered in the early 20th century and
refined in the late 20th century.
Modern analytical chemistry is dominated by instrumental analysis. Many
analytical chemists focus on a single type of instrument. Academics tend
to either focus on new applications and discoveries or on new methods of
analysis. The discovery of a chemical present in blood that increases the
risk of cancer would be a discovery that an analytical chemist might be
involved in.
Analytical chemistry is the study of the separation, identification, and
quantification of the chemical components of natural and artificial
materials.[1] Qualitative analysis gives an indication of the identity of the
chemical species in the sample, and quantitative analysis determines the
amount of certain components in the substance. The separation of
components is often performed prior to analysis.
Analytical methods can be separated into classical and instrumental
Classical methods (also known as wet chemistry methods) use separations

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such as precipitation, extraction, and distillation and qualitative analysis by
color, odor, or melting point. Classical quantitative analysis is achieved by
measurement of weight or volume. Instrumental methods use an apparatus
to measure physical quantities of the analyte such as light absorption,
fluorescence, or conductivity. The separation of materials is accomplished
using chromatography, electrophoresis or field flow fractionation methods.
Analytical chemistry is also focused on improvements in experimental
design, chemometrics, and the creation of new measurement tools to
provide better chemical information. Analytical chemistry has applications
in forensics, bioanalysis, clinical analysis, environmental analysis, and
materials analysis.
.

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Instrumental chemistry
Instrumental analysis is a field of analytical chemistry that investigates
analyses using scientific instruments.
The instrumental methods of chemical analysis are divided into categories
according to the property of the analyte that is to be measured. Many of the
methods can be used for both qualitative and quantitative analysis. The
major categories of instrumental methods are the spectral,
electroanalytical, and separatory.
The presence of many chemical substances can often be found by their
response to some external signal. The magnitude of this response is
proportional to the amount of substance present. Because electronic
equipment is often necessary to generate the external signal and/or to detect
the chemical response, these methods of quantitative analysis are called
instrumental methods. Instrumental methods are indirect, so the detecting
instrument requires calibration to measure the response initially from a
sample with a known concentration of analyte. This is necessary to relate
the response, which is often electrical, to the quantity of chemical
substance. Standard solutions, containing known amounts of analyte, are
first studied to calibrate the measuring instrument.
The type of instrumental method used for quantitative analysis varies with
the nature of the substance being analyzed and with the amount of analyte
thought to be present. While classical analytical methods are suitable for
major amounts of analyte present in a sample, 1% or greater, instrumental
methods are generally employed for amounts of analyte which may be less
than 1% of the sample's total mass. Modern instrumental techniques are
capable of analyzing the presence of a component which can comprise
0.0001% or less of its mass.
.

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Review
Atomic Absorption Spectrometric
Atomic Absorption Spectroscopy is an instrumental analysis technique for
rapid trace metal analysis.It is based on element specific wavelength light
absorption by ground state atoms in the flame or electrothermal graphite
furnace.
It finds immense applications in the analysis for trace metals in soils, lakes,
rivers, oceans, and drinking water, pharmaceuticals, foods and beverages,
geological and mineralogical samples, petroleum products, biological
fluids and specimens and forensic analysis. It is common to get results in
ppm levels and a higher sensitivity of ppb levels when we using graphite
furnace atomisation.
Uses of Atomic Absorption Spectroscopy
Atomic Absorption Spectroscopy provides cost-effective viable solutions
for the analysis of trace amounts of metals in the entire range of natural and
manmade materials such as Geological samples, Environmental samples,
Biological Specimens, Agricultural produce and soils, Pharmaceuticals,
Foods and Drinking water.
The technique affords advantages of speed, sensitivity and precision over
the classical gravimetric methods. Introduction of accessories such as
graphite furnace, flow injection analysis and improvements
in the suppression of matrix interferences have further contributed to
improvement in sensitivity and selectivity of analytes in complex matrices.
Atomic Absorption Spectroscopy applications in the field of environment,
drinking water, mining and mineralogy, oceanographic studies, soils,
pharmaceuticals, foods, toys, forensic investigations are of great
significance.

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The list is endless and presence or absence of trace metals is a factor that
cannot be overlooked for evaluation of characteristics of materials or
concerns regarding human health and safety.
The chemical techniques used for the analysis of trace metals have evolved
from simple gravimetric methods to highly sophisticated time saving
instrumental techniques. Atomic Absorption Spectroscopy is a popular
technique which involves moderate investment and affordable operational
cost.
These features coupled with a high degree of accuracy and precision of
results has contributed to the widespread presence of atomic absorption
spectrometers in college laboratories, industrial laboratories and regulatory
body laboratories across the world.
Principle of Atomic Absorption Spectroscopy
Atomic absorption spectroscopy (AAS) is based upon the principle that
free atoms in the ground state can absorb light of a certain wavelength.
These very specific wavelengths give the technique excellent specificity
and detection limits in the AAS analysis. Absorption for each element is
specific, no other elements absorb this wavelength. Typical applications of
AAS include –
 Quantitative metal concentrations in solution
 Analysis of lead in paint
 Monitoring of trace metals in industrial effluent streams
 Trace elements in product/raw materials along with ICP-MS
 Analysis of additives and purity in steels and other metal alloys
 Analysis of low-level contaminants
Types of Atomic Absorption Spectroscopy
Nowadays, Atomic Absorption Spectrometry (AAS) systems are
comparatively inexpensive instruments. Some also predict multi(few)-
element capability. There are various types of AAS – Flame (F AAS), Cold

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vapour (CV AAS), Hydride-generating (HG AAS), and Graphite furnace
(GF-AAS) systems.
Instrumentation of AAS
Atomizer
The sample must be atomized first in order to be studied. Atomization is
an important step in AAS as aids in determining the sensitivity of the
reading. An effective atomizer creates a large number of homogenous free
atoms. Though there are many types of atomizers present, only two are
commonly used: Flame and Electrothermal atomizers.
Radiation Source
There is a radiation source which irradiates the atomized sample. The
sample absorbs some of the radiation, and the rest passes through the
spectrometer to a detector. Radiation sources are of two categories: Line
sources and Continuum sources. Line sources excite the analyte and thus
emit its own line spectrum. Continuum sources have radiation that spreads
out over a wider range of wavelengths.
Spectrometer
Spectrometers are used to differentiate between various types of
wavelengths of light before they pass to the detector. The spectrometer in
AAS can be either single-beam or double-beam.
Single-beam spectrometers require radiation to pass directly through the
atomized sample. Whereas, double-beam spectrometers require two beams
of light – one beam that passes directly through the sample, and another
that does not pass through the sample at all.

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Atomic Absorption Spectrometric Analysis of Fluoroquinolones
Ciprofloxacin, Amoxicillin and Diclofenac Sodium
Ciprofloxacin, chemically 1,4-dihydro-1-cyclopropyl-6-fluoro-4-oxo-7-
(1-piperazinyl)-3-quinolinecarboxylic acid (Fig. 1a), is a quinolone
antibiotic drug with a broad spectrum of activity against a variety of gram
positive and gram-negative bacteria.
Amoxycillin (Fig. 1b) is the only phenolic penicillin used as an
antibacterial drug. It is a moderate-spectrum β-lactam antibiotic used to
treat bacterial infections caused by suscept ible microorganisms.
Diclofenac sodium is described as the salt of 2-[(2,6-
dichlorophenyl)amino]benzene acetic acid (Fig. 1c).

Figure 1 Molecular structures of ciprofloxacin (a), amoxycillin (b) and diclofenac

sodium (c).
A highly sensitive, simple and rapid atomic absorption spectrometric
method for application in quality control analysis. This method is based on
the reduction of iron(III) by the investigated drugs (ciprofloxacin,
amoxycillin and diclofenac sodium). The excess of iron(III) was extracted

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into diethyl ether22 and then iron(II) in aqueous layer was determined by
AAS. The reaction method is simple since it is a single step process.
Procedure
Six 2.5 ml standard solutions of ciprofloxacin (25–400 ng ml-1),
amoxycillin (50–500 ng ml-1) or diclofenac sodium (60–600 ng ml-1) were
pipetted into a series of 10 ml volumetric flasks. To each flask, 1.5 ml of
1.0×10-5 M ferric sulphate was added. The mixtures were heated using a
boiling water bath for 10 min (100 °C). After cooling, 4.0 ml of 12 M HCl
was added and the excess iron(III) was extracted with three portions of 10
ml of diethyl ether using a separatory funnel. Then the aqueous layer
containing iron(II) was aspirated into an air–acetylene flame. The
absorbance of iron(II) was measured at 302.1 nm, and the iron
concentration was determined from a previously constructed calibration
curve.
Results
It was found that ciprofloxacin, amoxycillin and diclofenac sodium were
very weak reducing agents at room temperature. The oxidation of these
compounds was non-quantitative, slow and time-consuming. However, on
heating at 100 °C using a boiling water bath, the given drugs immediately
reduced iron(III) to iron(II) in amounts which corresponded to the
concentration of the drugs. The amount of iron(II) was determined by AAS.
Comparison of the proposed and official method using the t-test and f-test
(p < 0.05) showed the high accuracy and precision of the AAS method. The
results obtained by the proposed and official method23 are summarized in
Table I.

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 Table 1 Assay of commercial tablet formulations by the AAS method and the
official method

The intraday, interday precisions and recoveries were tested (Table II).
These data indicate that the method was reproducible within and between
days. The mean percentage recovery ranged from 95 to 103 % (RSD < 6.1
%).
Table 2 Intraday and interday assay of ciprofloxacin, amoxycillin and diclofenac
sodium

The effects of the presence of common excipients (dextrose, glucose,

saccharine sodium, starch, talc and magnesium stearate) were tested (Table
III). There were no significant interferences due to the presence of any of
these excipients.
Effect of temperature
The effect of the temperature was investigated by heating the reaction
mixtures of these drugs in a water bath at different temperatures for 10 min.
The absorbance was found to increase with increasing reaction
temperature. The maximum absorbance was observed at 100 °C, as shown
in Fig. 2. The time of heating is important to ensure complete reaction.

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Different heating time intervals were investigated at a constant temperature
(100 °C); 10 min was found to be the optimal interval time to achieve
complete reaction, as is shown in Fig. 3.

Figure 2 Effect of temperature on the oxidation reactions of the drugs,

ciprofloxacin 0.4 µg ml-1, amoxycillin 0.5 µg ml-1, and diclofenac sodium 0.3 µg
ml-1.

Figure 3 Effect of heating time on the oxidation reactions of the drugs,

ciprofloxacin 0.4 µg ml-1, amoxycillin 0.5 µg ml-1, and diclofenac sodium 0.3 µg
ml-1.
Effect of pH
The effect of pH on the response of the oxidation reactions was determined
by recording the absorption of ciprofloxacin (400 ng ml−1), amoxycillin
(500 ng ml−1) and diclofenac sodium (300 ng ml−1) at different pH values.
The absorption vs. pH graph, Fig. 4, showed the absorbance was almost

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independent of pH in the range 1.0–2.5 for ciprofloxacin, amoxycillin and
diclofenac sodium.

Figure 4 Effect of pH on the oxidation reactions of the drugs, ciprofloxacin 0.4 µg

ml-1, amoxycillin 0.5 µg ml-1, and diclofenac sodium 0.3 µg ml-1.
Iron (III)/Drug mole ratio

Figure 5 Effect of iron(III) concentration on the drugs, ciprofloxacin 0.4 µg ml-1,

amoxycillin 0.5 µg ml-1, and diclofenac sodium 0.3 µg ml-1.

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Conclusion
The AAS method is highly sensitive and, moreover, it can be used for
routine analysis of the investigated drugs in raw materials and
pharmaceutical formulations. The method is simple and rapid since it is a
single step process. The statistical parameters and the recovery tests data
clearly indicate the reproducibility and accuracy of the method. The results
demonstrate that the method has an equivalent accuracy and precision as
the official methods, as found from the t and f-tests.

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References
1. Chemlstry and The Llvlng Organism, Fourth Edition Molly M.
Bloomfield. Wiley: New York. NY, 1987. xviii + 610 + 1-10 pp.
Figs. and tables.
2. Essentials of Quantitative Analysis C. David Wesf. McGraw-Hill:
New York, NY, 1987. xx + 458 pp. Figs. and tables.
3. Saleh, G.A., 1997. Spectropotometric determination of
fluoroquinolones. Bull. Phrm. Sci., Assuit University, 20: 27.
4. Park, H., C. Oh, H. Lee, J. Lee, K. Yang and K. Bark, 2002.
Spectroscopic Properties of fluoroquinolone antibiotics in water-
methanol and water acetonitrile mixed solvents. Photochem.
Photobiol. 75: 237-48
5. M. A. Abulkibash, M. S. Sultan, M. A. Al-Olyan, M. S. Al-
Ghannam, Talanta 61 (2003) 239
6. M. A. Gonzalez, F. Uribe, S. D. Moisen, A. P. Fuster, A. Selen, P.
G. Welling, B. Painter, Antimicrob. Agents Chemother. 26 (1984)
741
7. X. Du, C. Li, K. H. Sun, H. C. Nightingale, H. Charles, P. D.
Nicolau, J. Pharm. Biomed Anal. 39 (2005) 648
8. G. H. Ragab, A. S. Amin, Spectrochim. Acta 60 (2004) 973
9. B. Kanakapura, P. H. Chandrashekar, Ind. Sci. Asia 29 (2003) 141
10. A. I. Vogel, Vogel's Textbook of Quantitative Inorganic Analysis,
4th Ed., Longman Press, New York, 1978, p.157

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