ANALYSIS 501722
FACULTY OF PHARMACY &MEDICAL SCIENCE
Petra University
Dr. Wael Abu Dayyih
2012
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An Introduction to Pharmaceutical
Instrumental Methods
Dr. WAEL ABU DAYYIH
2011
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wabudayyih@uop.edu.jo
Office Hours: Sun.Tue, The. (11-12.00)
And also by individual student appointments.
(Office hours are subject to change with any changes being announced in class)
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1) Overview Pharmaceutical analysis
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2) Analysis and pharmaceutical quality control
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3) Evaluation of data in pharmaceutical analysis
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4) Control of the quality of analytical methods
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5) Physical and chemical properties of drug molecule
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6) Spectral signals/Uv-Spectrometer
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7) Spectral signals/IR-Spectrometer
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8) Separation signal/Chromatography- HPLC
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9) Electrical signals/ Potentiometry and ISE
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10) Thermal signal /DSC
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Course Text:
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Principles of Instrumental Analysis (6th Edition) by Skoog,
Holler and Crouch, published by Thomson Brooks/Cole.
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Pharmaceutical Analysis 2ed edition by David Watson
published by Elsevier Churchill Livingstone
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Note: Supplementary course material,
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Along with class handouts, will be provided in class or on
the web and will be announced in-class.
Evaluation:
 15 marks first exam.
 15 marks second exam
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20 marks Lab.
 10marks activity
 40 marks final
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Scale
50-54
60-64
70-74
80-84
90-94
D
CC+
B
A-
55-59
65-69
75-79
85-89
95-100
First exam( )
 Second exam (
D+
C
BB+
A
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)
Introduction
 Analytical Chemistry deals with methods for determining the
chemical composition of samples of matter.
 A qualitative method yields information about the identity of
atomic or molecular species or the functional groups in the
sample; a quantitative method, in contrast, provides
numerical information as to the relative amount of one or
more of these components.
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Classification of Analytical Methods
 Analytical methods are often classified as being either
classical or instrumental.
 This classification is largely historical with classical methods,
sometimes called wet-chemical methods, preceding
instrumental methods by a century or more.
 In the early years of chemistry, most analyses were carried
out by separating components of interest in a sample by
precipitation, extraction, or distillation.
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For qualititative analyses, the separated components were then treated with
reagents that yielded products that could be recognized by their colors, boiling
points or melting points, their solubility in a series of solvents, their odors,
their optical activities, or their refractive indexes.
For quantitative analyses, the amount of analyte was determined by
gravimetric or by titrimetric measurement.
In the former, the mass of the analyte or some compound produced from the
analyte was determined.
In Titrimetric procedures: the volume or mass of a standard reagent
required to react completely with the analyte was measured.
These classical methods for separating and determining analyte still fund use
in many laboratories. The extent of their general application is, however,
decreasing with the passage of time.
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Instrumental Methods
 In the mid-1930s, or somewhat before, chemists began to
exploit phenomena other than those described in the
previous section for solving analytical properties of analytes –
such as conductivity, electrode potential, light absorption or
emission, mass-to-charge ratio, and fluorescence – began to
be employed for quantitative analysis of a variety of
ignorance, organic, and biochemical analytes.
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Instrumental Methods
 Furthermore, highly efficient chromatographic separation
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techniques began to supplant distillation, extraction, and
precipitation for the separation of components of complex
mixtures prior to their qualitative or quantitative determination.
These newer methods for separating and determining chemical
species are known collectively as instrumental methods of analysis.
Many of the phenomena upon which instrumental methods are
based have been known for a century or more.
Their application by most chemists, however, was delayed by a
lack of reliable and simple instrumentation.
In fact, the growth of the modern instrumental methods of
analysis has paralleled the development of the electronic and
computer industries
Types of Instrumental Methods
 For this discussion, it is convenient to describe physical
properties that are useful for qualitative or quantitative
analysis as analytical signals.
 Table 1-1 lists most of the analytical signals that are currently
used for instrumental analysis.
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analytical signals.
 Note that the first six involve electromagnetic radiation.
 The first, the radiant signal is produced by the analyte; the next five
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signals involve changes in a beam of radiation brought about by its
passage into the sample.
Four electrical signals then follow, finally, for miscellaneous signals are
grouped together. These include mass-to-charge ratio, reaction rate,
thermal signals, and radioactivity.
The second column in Table 1.1 lists the names of instrumental
methods that are based upon the various analytical signals.
It should be understood that beyond their chronology, few features
distinguish instrumental methods from their classical counterparts.
Some instrumental techniques are more sensitive than classical
techniques, but others are not.
With certain combinations of elements or compounds, an
instrumental method may be more selective; with others, a
gravimetric or volumetric approach may suffer less from interference
Signals Employed in Instrumental Methods
 (X-ray, UV, visible, electron, Auger);
fluorescence, phosphorescence, and
luminescence
(X-ray, UV, and visible)
 Emission of radiation
 Spectrophotometry and photometry (X-ray,
UV, visible, IR); photoacoustic spectroscopy;
nuclear magnetic resonance and electron spin
resonance spectroscopy
 Absorption of radiation
 Turbidimetry; nephelometry; Raman
spectroscopy
 Scattering of radiation
 Refractometry; interferometry
 Refraction of radiation
 X-Ray and electron diffraction methods
 Diffraction of radiation
 Polarimetry; optical rotary dispersion; circular
dichroism
 Rotation of radiation
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o Potentiometry; chronopotentiometry
 Electrical potential
o Coulometry
 Electrical charge
o Polarography; amperometry
 Electrical current
o Conductometry
 Electrical resistance
o Mass spectrometry
 Mass-to-charge ratio
o Kinetic methods
 Rate of reaction
o Thermal conductivity and enthalpy
 Thermal properties
o Activation and isotope dilution methods
 Radioactivity
N0TE…..!!!
 Generalization on the basis of accuracy, convenience, or expenditure of time are
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equally difficult to draw.
is it necessary true that instrumental procedures employ more sophisticated or
more costly apparatus; indeed, the modern electronic analytical balance used
for gravimetric determinations is a more complex and refined instrument than
some of those used in the other methods listed in Table 1-1.
As mentioned earlier, in addition to the numerous methods listed in the second
column of Table 1-1, there exists a group of instrumental procedures that are
employed for separation and resolution of closely related compounds,
The majority of these procedures are based upon chromatography.
One of the signals listed in Table 1-1 is ordinarily used to complete the analysis
following chromatographic separations.
Thermal conductivity, ultraviolet and infrared absorption, refractive index, and
electrical conductance have been employed for this purpose.
This text deals with the principles, the applications, and the performance
characteristics of the instrumental methods listed in Table 1-1 and of
chromatographic separation procedures as well.
No space is devoted to the classical methods, the assumption being that the
reader will have encountered these techniques in earlier studies
Instrument for Analysis
 In the broadest sense, and instrument for chemical analysis converts an analytical signal that is
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usually not directly detectable and understandable by a human to a form that is.
Thus, an analytical instrument can be viewed as a communication device between the systems
under study and the scientist.
An instrument for chemical analysis is general made up of just four basic components.
As shown in Figure, these components include a signal generator, an input transducer
(called a detector), a signal processor, and an output transducer or readout device.
A general description of these components follows
A signal generator produces a signal that reflects the presence and usually the concentration of
the analyte.
In many instances, the signal generator is simply a compound or iron generated from the
analyte itself.
For atomic emission analysis, the signal generator is excited atoms or ions of the analyte that
emit photo of radiation.
For a pH determination, the signal is the hydrogen ion activity of a solution containing the
sample.
In many other instruments, however, the signal generator for an instrument for an infrared
absorption analysis includes, in addition to the sample, a sound of infrared radiation, a
monochromator, a beam chopper and splitter, a radiation attenuator, and a sample holder.
Some Examples of Instrument Components TAB.1.2
INSTRUMENT
SIGNAL
GENERATO
R
ANALYTICAL
SIGNAL
INPUT
TRANSDUCER
TRANSDU
CER
SIGNAL
SIGNAL
PROCESSOR
READOUT
PHOTOMETER
TUNGSTEN
LAMP,GLASS
FILTER,
SAMPLE
ATTENUTED
LIGHT BEAM
PHOTOCELL
ELECTRICAL
CURENT
NONE
CURRENT
METER
ATOMIC
EMISSION SPECT.
FLAM
MONO-
UV OR VISIBLE
RADIATION
PHOTOMULTIPLIER
AMPLIFIER
TUBE
ELECTRICAL
POTENTIAL
DEMODULATOR
CHART
RECORDER
CHROMATOR
,SAMPLE
COULOMETER
DC SOURCE
SAMPLE
CELL
CURRENT
ELECTRODES
ELECTRICAL
AMPLIFIER
CURRENT
CHART
RECORDER
pH METER
SOLUTION
SAMPLE
HYDROGEN
ION ACTIVITY
GLASS CALOMEL
ELECTRODES
ELECTRICAL
POTENTIAL
AMPLIFIER
DIGITIZER
DIGITAL UNIT
X-RAY POWDER
DIFFRACTOMETER
X-RAY TUBE
SAMPLE
DIFFRACTED
RADIATION
PHOTOGRAPHIC
FILM
LATENT
IMAGE
CHEMICAL
DEVELOPER
BLACK IMAGES
ON FILM
COLOR
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COMPARATOR
SUNLIGHT
SAMPLE
COLOR
EYE
OPTIC
NERVE
SIGNAL
BRAIN
VISUALCOLO
R RESPONSE
Detectors (Input Transducers)
 A transducer is a device that converts one kind of energy
(or signal) to another. Examples include a themocouple, which
converts a radiant heat signal into an electric voltage; a photocell,
which converts light into an electric current; or the beam of a balance,
which converts a mass imbalance into a displacement of the beam of a
balance from the horizontal.
 Transducers that act on a chemical signal are called
detectors.
 Most detectors convert analytical signals to an electric voltage or
current that is readily amplified or modified to drive a readout device.
 Note, however, that the last two detectors listed in Table 1-2 produce
non-electric signals
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Signal Processors
 The signal processor modifies that transduced
signal from the detector in such a way as to make it
more convenient for operation of the readout
device.
 Perhaps the most common modification is amplification – a
process in which the signal is multiplied by a constant greater
than unity.
 In a two-pan balance, the signal processor is a pointer on a
scale whose displacement is considerably greater than the
displacement of the beam itself.
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AMPLIFIER
 Amplification by a photographic film is enormous; here, a
single photon may produce as many as 1012 silver atoms.
 Electric signals, of course, are readily amplified by a factor of
1012 or more.
 A variety of other modifications are also commonly carried
out on electric signals.
 In addition to amplification, signals are often filtered to
reduce noise,
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Readout Devices
 A readout device is a transducer that converts a
processed signal that is understandable by a human
observer. Usually, the transducer signal takes the form of
the position of a pointer on a meter scale, the output of a
cathode-ray tube. In some instances, the readout device give
an analyte concentration directly.
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Circuits and Electrical Devices in
Instruments
 The detector in most modern analytical
instruments converts the analytical signal
to an electric one, which is processed in
various ways and them displayed by a
readout device.
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Microprocessors and Computers in
Instruments
 Modern analytical instruments generally employ
one or more sophisticated electronic devices – such
as operational amplifiers, integrated circuits,
analog-to-digital and digital-to-analog converters,
counters, microprocessors, and computers.
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SELECTING AND ANALYTICAL METHOD
 DEFINING THE PROBLEM
 In order to select an analytical method intelligently, it is essential
to define clearly the nature of the analytical problem. Such a
definition requires answers to the following questions:
 What accuracy and precision are required?
 How much sample is available?
 What is the concentration range of the analyte?
 What components of the sample will cause interference?
 What are the physical and chemical properties of the sample
matrix?
 How many samples are to be analyzed?
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SELECTING AND ANALYTICAL METHOD
 The answer to question 1 is to of vital importance because it
determines how much time and care will be needed for the
analysis. The answers to questions 2 and 3 determine how
sensitive the method must be and how wide a range of
concentrations must be accommodated. The answer to
question 4 determines the selectivity required of the method.
The answers to question 5 are important because some
analytical methods in Table 1-1 are applicable to solutions
(usually aqueous) of the analyte. Others are more easily
applied to gaseous samples, whereas still others are suited to
the direct analysis of solids.
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SELECTING AND ANALYTICAL METHOD
 The number of samples to be analyzed question 6 is also an
important consideration from the economic standpoint. If
this number is large, considerable time and money can be
spent on instrumentation, method development, and
calibration. Furthermore, if the number is to be large, a
method should be chosen that requires the least operator
time per sample. On the other hand, if only a few samples
are to be analyzed, a simpler but more time-consuming
method that requires little or no preliminary work is often
the wiser choice.
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Conclusion
 With answers to the foregoing six questions, a method can
then be chosen – provided the performance characteristics of
the various instrumental methods shown in Table 1-1 are
known.
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factors that should be considered when choosing an
available method
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
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The type of available instrument
The experience of the analysis is using the instrument
The excepted concentration range within with the sample falls
The required precession and accuracy of assay
The possible interferes f the assay
The # of the samples that must assayed with the instrument
The rate at which must the result be obtained
The expense of each assay while using the instrument
Lists quantitative performance criteria of instruments, criteria that can be
used to decide whether factors that should be considered in glossary.
Criteria
 1. Precision-------------------------
 3. Sensitivity-----------------------
Figure of Merit
 1.Absolute standard deviation,
relative standard deviation,
coefficient of variation, variance
 2.Absolute systematic, error,
relative systematic error
 3.Calibration sensitivity, analytical
sensitivity
 4. Detection limit-----------------
 4.Blank plus three times standard
 2. Bias-------------------------------
 5. Concentration range----------
 6. Selectivity------------------------
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deviation of a blank
 5.Concentration limit of
quantitation (LOQ) to
concentration limit of linearity
(LOL)
 6.Coefficient of selectivity
Characteristics to Be Considered in
Method Choice
 Speed
 Ease and Convenience
 Skill required of operator
 Cost and availability of equipment
 Per-sample cost
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Analysis and Pharmaceutical Quality
Control
Types of Analysis:
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 1.
Qualitative Analysis:
 2.
Quantitative Analysis:
Quantitative Analysis
 How much of a compound is there present?
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Most Useful Methods:
a. NMR: good at percent concentration levels not at trace
levels.
b. IR: needs high concentrations and mostly done for liquid
samples.
c. UV: used especially for unsaturated compounds in natural
products, not for trace levels but for percent levels, it is subject to
spectral overlap and interference from other compounds in the
sample but can be overcome by certain techniques.
Other: Thermal analysis, X-ray diffraction.
Qualitative Analysis
 WHAT ARE THE COMPONENTES PRESENTS ?
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Most useful methods:
a. NMR: identifies type of H, C in organic compounds and
revels the position of functional groups relative to each other.
b. IR: indicates for functional groups and hetero elements and
distinguishes between aliphatic and aromatic systems
c. MS: useful to identify very high molecular weight and
identifies functional groups and structure of the compound. It
also confirms the presence of hetero elements.
d. UV: Determination of unsaturated compounds especially
aromatics and other functional groups.
Other: X-ray fluorescence and X-ray diffraction.
Classification of Analytical Methods
 A.
Classical Analysis:
 Titration  Quantitative
 While reacting with a reagent to give a product with a color,
boiling point or melting point, solubility and refractive
index.
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Classification of Analytical Methods
 B.
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Instrumental Analysis:
I. based on measurement of a certain physical property (nonSeperational technique)
Spectroscopy, Techniques: (Fluorescence, IR, NMR, UV, MS)
Electrochemical: Potentiometry
II. Seperational Techniques
Chromatographic techniques: (HPLC, GC, CE, TLC)
Crystalization
Extraction
Distillation
Areas of Applying Analysis
 Fundamentals research
 Product development
 Product Quality control
 Monitoring and control of pollutants
 Assay
 Medical and chemical studies
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Application in Pharmaceutical Industry
 Design and testing of new drugs "Combinatorial chemistry”
 QC/QA of raw materials, intermediates and formulated
products "Intermediate Analysis"
 Stability testing "Accelerated degradation"
 PK ( pharmacokinetic.)and metabolism studies
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Sampling
 The most important step in analysis:
 It should be representative, sufficient for all analysis to be carried
out comfortably
 Pre-treatment:
 To decrease interference
 bring the analyte into a desired range of concentration by
dissolution, fusion, separation, dilution concentration, derivatization
 Control of the chemical environment, controlling the
atmosphere which the sample is exposed to and controlling
different instrumental parameters
 Sampling different sample types:
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sample types
 Gas Samples:
 Single spot sample taken by balloon or syring after sample
has been stirred well with a fan pr paddle
 Several composite samples of gas can be bleeding gas slowly
in a suitable container over a period of time
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sample types
 Liquid Samples
 Stir before sampling
 Sample remote from sources of contamination
 River or sea samples should be taken at points where
contamination is minimal, taken at several depths and
distances from shore.
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sample types
 Solid Samples: Most difficult to handle, can't be mixed well
 take portions from different parts and mix them and
make a representative sample.
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sample types
 Metal Sample:
 When a molten metal solidifies the purist solidifies first
 The core contains most impurities so ground a representative
cross section of the sample,
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Bulk Samples: as coal, soil,…
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Storage of Sample
 Container should be clean and air tight
 Liquid and gas can be stored in plastic containers
 Solid samples should be sealed
 All samples should be stored in room not too hot or too
humid
 Avoid storage for long times (decomposition, evaporation)
special avoid glass bottles
 Advised freeze sample
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Steps in an Analysis
1. Definition of the Problem:
This is the first stage in planning the analysis, and can be considered as
the process of deciding what analytical information is needed.
2. Choice of the Technique:
Having decided what information is required, you next decide in detail
how are you going to obtain it.
3. Sampling:
The process of selecting and removing a small representative part of a
whole on which you will perform the analysis.
4. Sample pre-treatment and separation:
Only in the simplest of situations can the sample be analyzed without
some form of pre-treatment to convert it into a form suitable for
analysis.
This pre-treatment may or may not involve separation.
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Steps in an Analysis
5. Qualitative Analysis:
Test on the (pre-treated) sample. (Under controlled conditions)
Tests on reference materials for comparison. (Under controlled
conditions).
Interpretation of the tests.
6. Quantitative Analysis: (Needs more controlled conditions)
Calibration – obtaining raw analytical data from suitably prepared standards.
Measurements – obtaining the raw analytical data from measurements on the
(pre-treated) sample. Maybe recalibration due to not valid for a long time.
Evaluation – calculating a meaningful analytical result from the raw data.7
7. Action:
The result is used to make a decision as to what action to take in
relation to the original problem.
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NEXT CHAPTER
 CONTROLE OF QUALITY OF ANALYTICAL
METHODS
 VALIDATION METHODS
Good Luck
Dr. WAEL ABU DAYYIH
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