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CHAPTER 1
PART I
INTRODUCTION TO DRUGS
PART II
INTRODUCTION TO SPECTROPHOTOMETRY
PART III
INTRODUCTION TO X-RAY CRYSTALLOGRAPHY
1
PART I
INTRODUCTION TO DRUGS
1. I. 1 INTRODUCTION
1. I. 2 DRUGS AND FORMULATIONS
2
1.I. 1 INTRODUCTION
Medicines have become the part and parcel of mankind. Drugs, in the form
of plant products and minerals have been used from times immemorial. Primitive
man discovered appropriate cures for various ailments through trial and error by
chewing plant products such as roots, bark, leaves and fruits, and this information
was passed on to subsequent generations by word of mouth. Drugs can leave
residual effects after administration has ceased. Based on the origin of a medicinal
product the system is labeled as Ayurvedic, Unani, Homeopathic etc. The widely
used medicines belong to allopathic systems. Irrespective of the origin of the
medicine, they are judged and accepted only when it is quality assured; having
minimum side effects and the formulation is well defined. This requires analysis of
the drugs and an authencity label is fixed. Many drugs also have a definite life. It is
therefore necessary that not only the effective part of formulations but also others
present as ingredients must be subjected to analysis. Several analytical approaches
are available; each approach depends on the nature of the drug and its activity. Thus
analytical techniques in drug analysis become selective.
Considering the above aspects several analytical methods have been
explored in this work pertaining to the drug formulations like artemisinin and its
derivatives, glucosamine hydrochloride and sulphate, carvedilol and nevirapine.
Before moving to our present investigations, it is necessary to have an overall view
of the work already done.
1. I. 2 DRUGS AND FORMULATIONS
Any substance that is carefully used for diagnosis, cure and prevention for
altering the structure and function of the body is called drug1. In the modern era,
drugs play an important role in the progress of human civilization. While primitive
man depended mainly on plant product and metal salts to cure diseases, modern man
uses a wide range of synthetic organic compounds and biotechnology- derived
antibiotics, vaccines, etc. There are many important stages before a compound is
used as a drug. The three important stages in the use of a drug as a medicine, i.e., the
conversion of a drug into a formulation are
3
i. The discovery of the drug.
ii. The manufacture of the drug in bulk form.
iii. The formulation of a drug into different dosage forms like tablet, capsule,
injection, syrup etc.
First stage is the drug discovery, where the compounds are screened for
biological activities. Second stage is the manufacture of the drug using well-
understood chemistry and adapting safe and proper manufacturing and analytical
practices and the third stage is the formulation of the drug in a convenient dosage.
Chemists play an important role in pharmaceutical research, as they synthesize,
purify and analyze the drugs. The study of conversion of drugs into medicine and its
manufacture, stability and the effectiveness of the drug dosage form is termed as
pharmaceutics.
The preparation, chemical and physical composition, reactive nature,
geometry, influence on an organism, quality control methods, storage conditions and
like which are pre-requisites in the study of drugs fall under pharmaceutical
chemistry, a potential field, based on the general laws of chemistry 2-7. The family of
drug could be either chemotherapeutic or pharmacodynamic agents.
Pharmacodynamic are a group of drugs, which depress or stimulate various
functions of the body, providing some relief by mitigating any abnormality in the
body. Though they are not likely to cure the diseases, they may provide temporary
relief. Depressants, stimulants towards central nervous system, adrenergic, blocking,
cholinergic, cardiovascular, diuretics, antihistaminic, anticoagulating agents belong
to this group. These have no action on infective organisms.
Chemotherapeutic agents are selectively more toxic to the invading
organisms. They cause no harm to the host. Antimalarials, antibacterials,
antiprotozoals, organometallic agents belong to this group.
Every bulk drug and corresponding pharmaceutical formulations have to
follow the set standards by each country through legislation8. Several
pharmacopoeia publications do furnish these regulations 9-12. Pharmaceutical
analysis13,14 deals not only with drugs and their formulations but also with their
4
precursors. However, degree of purity and the quality of medicament is a must. The
quality of a drug is decided only after its authenticity is tested, that too in the drug
and its formulations. Whatever may be the application, quality is paramount as it is
more vital in the field of medicine as the target is life15. One has to consider the
process of production of a drug and meticulously prevent impurities and toxic
elements which may peep in.
The above requirement necessitates that the whole operation from raw
material to the final product in the form of a drug or formulations must go through a
quality control unit. This hinges on good laboratory practice. Here the role of an
analyst who can do both qualitative and quantitative determination of not only the
raw material but also the drug in bulk and their pharmaceutical formulations
becomes invaluable. The acceptance of these life saving products in the market
needs a quality assurance stamp. One has to consider the brand name also. This is
possible only through an acceptable analytical approach.
The selection as a drug depends upon one or two types of control actions
independent or together. For a product of single entity having high purity, analytical
data will suffice. However, invariably the formulations are physical mixtures of
several potent drugs. The growth of pharmaceutical industry, increase in the number
and variety of drugs and availability of sophisticated instruments has paved way for
rapid progress in providing simple analytical procedures for the analysis of complex
formulations also.
The time tested assays of medicinal products are no doubt still dependable.
The availability of new techniques with improved equipments has made the latest
techniques attractive. The precision, accuracy, time and economy are a prime factor
here. Also many pharmaceuticals need not be analyzed by the same procedure. The
latest knowledge has thrown open the possibility of adopting unique techniques for
assaying a single drug alone or a number of drugs in a formulation at one stroke.
Separation techniques, particularly chromatographic methods are valuable in
analysis of pharmaceuticals. Modern spectrophotometer which incorporates features
such as microprocessor control, diode array detector has become essential tools for
analysis. Assay methods based on absorption in the ultraviolet and visible region of
5
electromagnetic spectrum are used extensively. Some colorless substances required
to be analyzed are converted to a derivative having color, the intensity of color
measured at suitable wavelength and compared with that of known amount of
reference substance of known purity. Solvents used for dilution for UV-visible
spectrophotometric assay require special purification different from the requirement
for other uses. It is preferable that blanks are run on the solvent and reagents used to
obtain a correction for their inherent absorbances.
Several methods for the estimation of drugs are classified into physical,
chemical, physico-chemical and biological ones. Physical methods involve the study
of the physical properties such as solubility, transparency or degree of turbidity,
color density, specific gravity etc. Physico-chemical methods involve the study of
the physical phenomena that occurs as a result of chemical reactions16-18. These
include optical and chromatographic methods. The combination of mass
spectroscopy with gas chromatography is one of the most powerful tools available.
The chemical methods include the gravimetric and volumetric procedures which are
based on complex formation, redox reactions etc. Titration in non-aqueous media
and complexometry are also being used in pharmaceutical analysis.
The continuous growth of new drugs needs new methods for controlling the
quality. Modern pharmaceutical analytical techniques need the following
requirements.
i. minimal time for analysis
ii. analysis accuracy should satisfy the demands of pharmacopoeia
iii. analysis should be economical
iv. the selected method should be precise and selective
v. the above requirements are met by the physico-chemical methods of
analysis. An advantage is their universal nature that can be employed for
analyzing organic compounds with any diverse structure. Visible
spectrophotometry is generally preferred especially by small scale industries
as the cost of the equipment is less and the maintenance problems are
minimal.
6
PART II
INTRODUCTION TO SPECTROPHOTOMETRY
1. II. 1 SPECTROPHOTOMETRY
1. II. 2 DEVIATION FROM BEER’S LAW
1. II. 3 DEVELOPMENT OF METHODS
1. II. 4 CALIBRATION CURVE
1. II. 5 CHOICE OF WAVELENGTH
1. II. 6 SENSITIVITY OF SPECTROPHOTOMETRIC METHODS
1. II. 7 PRECISION AND ACCURACY
1. II. 8 DETECTION LIMIT
1. II. 9 QUANTITATION LIMIT
1. II. 10 COMPARISON OF RESULTS
1. II. 11 COLOR DEVELOPMENT
1. II. 12 CHOICE OF SOLVENT
1. II. 13 LIMITATIONS
1. II. 14 APPLICATIONS
7
1. II. 1 SPECTROPHOTOMETRY
In the past few decades, a number of elegant instrumental techniques were
reported which are rapid, selective and having a high degree of accuracy. Among
these, spectrophotometry is the most important method, which is widely used for
wide variety of materials. High accuracy, precision, sensitivity and the ease of
availability of spectrophotometer made this technique indispensable for the modern
analytical chemists.19 Besides, it offers the advantage of having calibration graphs
that are linear over a wide range when compared to other spectroscopic techniques.
A very extensive range of concentration of substances (10-2 – 10-8 M) may be
covered. Analytical chemistry plays an important role in the modern era, especially
in pharmaceutical industries, which rely upon both the quantitative and qualitative
chemical analysis. Techniques frequently employed in pharmaceutical analysis
include UV-Vis, AAS and IR. Titrimetric method is an important and still growing
area in the field of analytical chemistry due to its versatility, simplicity and rapidity.
The theory behind spectrophotometric methods lies on a simple relationship
between the color of the substance and its electronic structure. A molecule exhibits
absorption in the UV-Vis region when the radiation causes an electronic transition in
molecules containing chromophoric groups. In these techniques color is an
important criterion for the identification of constituents. The importance of colored
solution lies on the fact that the radiation absorbed is the characteristic of the
material responsible for absorption and can be determined quantitatively or
qualitatively. Nevertheless, a substance that is colorless or faintly colored may be
often determined by the addition of chromogenic reagent, imparting intensive color
to the species. The quantitative applicability of the absorption method is based on
the fact that the number of photons absorbed is directly proportional to the number
or concentration of atoms, ions or molecules.20
The art of identifying materials based on their color was probably the earliest
examples of qualitative molecular absorption spectrophotometry. Also the
recognition that color intensity can be the indicator of concentration was probably
the earliest application of molecular absorption spectrophotometry. Initially using
human eye as the detector and undispersed sunlight or artificial light as the light
source made the measurements. The introduction of optical filters, which isolates
8
specific frequencies of light, improved the accuracy and precision of the
measurements to some extent. Further improvement of the measurement came with
the use of prism and grating monochromator for wavelength isolation and also
photoelectric detectors, phototubes and photomultiplier tubes. Development of solid-
state microelectronics has now made available a wide range of detector type which
coupled with the computers, provide highly sophisticated electronic systems.
1. II. 2 DEVIATION FROM BEER’S LAW
From Beer’s law it follows that if we plot absorbance verses concentration, a
straight line passing through the origin should be obtained 21. It generally holds over
a wide range of concentrations if the structure of the colored ion or of the colored
non-electrolytes in the dissolved state does not change with concentration. The
presence of small amount of colorless electrolytes, which do not react chemically
with the colored components, normally does not affect the light absorption. Large
amount of electrolytes may result in a shift of maximum absorption and may also
change the value of the extinction coefficient.
When the colored species ionizes, dissociates or associates in solution,
Beer’s law will usually not be obeyed as the nature of species in solution will vary
with the concentration. The law does not hold well when the colored solute forms
complexes, the composition of which depends upon the concentration. Deviation
from Beer’s law may also occur when monochromatic light is not used.
1. II. 3 DEVELOPMENT OF METHOD
In developing a quantitative method for determining an unknown
concentration of a given substance by absorption spectrophotometry, the first step is
the selection of analytical wavelength at which measurements are to be carried out.
In order to enhance the sensitivity of the method and signal to noise ratio, the
wavelength of maximum absorbance is chosen as analytical wavelength. After
setting the analytical wavelength, the color developing reagent and the absorbing
product must be stable for a considerable period of time.
9
1. II. 4 CALIBRATION CURVE
The common method of using the spectrophotometer requires the
construction of a calibration curve for the constituents being determined. Calibration
is one of the most important steps in drug analysis. For this purpose, suitable
quantities of the constituents are taken and treated in exactly the same way as the
sample solution for the development of color, followed by the measurement of the
absorption at the optimum wavelength. The absorbance is then plotted against
concentration of he constituents. A straight line is obtained if Beer’s law is followed.
This calibration curve may then be used to determine the constituents under the
same conditions. The calibration curves needs checking at intervals.
1. II. 5 CHOICE OF WAVELENGTH
It is important to avoid making measurements in the region where the molar
absorptivity (ε) changes rapidly with the wavelength. In such a region even a small
error in setting the wavelength scale will result in a large apparent molar
absorptivity22. Therefore, it is necessary to select the wavelength corresponding to
maximum ε. Beer’s law will not be obeyed when the transmittance of the solution
increases continuously over the wavelength range covered by the light filter.
1. II. 6 SENSITIVITY OF SPECTROPHOTOMETRIC METHODS
Sensitivity is often described in terms of the molar absorptivity (ε, L mol-1
cm-1). The awareness of the sensitivity is very important in the determination of
pharmaceutical compounds. The objective numerical expression23-25 of the sensitivity
of spectrophotometric methods is the molar absorptivity (ε) at the wavelength (λ max)
of maximum absorbance of the colored species,
Molar absorptivity (ε) = A / c l
The sensitivity of spectrophotometric measurements depends on the
monochromaticity of the radiation. The molar absorptivity diminishes as the band-
width increases.
Savvin26 suggested a relation between sensitivity and molar absorptivity. He
suggested the following criteria for describing the sensitivity.
Low sensitivity, ε < 2 × 104 L mol-1 cm-1
Moderate sensitivity, ε = 2 - 6 × 104 L mol-1 cm-1
High sensitivity, ε > 6 × 104 L mol-1 cm-1
10
The molar absorptivity cannot exceed more than 1.5 × 105 L mol-1 cm-1,
according to quantum theory.
Other ways of specifying sensitivity are as specific absorptivity27 or
Sandell’s sensitivity28. In both the methods sensitivity is expressed in terms of
amount of analyte per unit volume of solution. Such an approach is perhaps more
convenient than using molar absorptivities as a basis of comparison. Sandell’s
sensitivity is the concentration of the analyte (µg mL-1) which will give an
absorbance of 0.001 in a cell of path length 1 cm and is expressed as µg cm-2.
Organic reagents with high molecular weights furnish maximum sensitivity if used
as chromogenic agents. Detection limits can be reduced to somewhat by solvent
selection because molar absorptivities depend on the solvent system. Another
technique used to increase the detection limit is to use indirect determinations,
where a stoichiometric gain in the number of chromophores may result or the newly
formed chromophore may have a higher molar absorptivity. Reaction rate methods
can sometimes have lower detection limits than do conventional spectrophotometric
measurements.
1. II. 7 PRECISION AND ACCURACY
Precision describes reproducibility of results where accuracy denotes the
nearness of a measurement to its accepted value. The accuracy and precision of
spectrophotometric method depends on three major factors, instrumental limitations,
chemical variables and operators’ skill. Instrumental limitations are often
determined by the quality of the instruments, optical, mechanical and electronic
systems. Chemical variables are determined by purity of standards, reagents and
chromophore stability, reaction rates, reaction stoichiometry, pH and temperature
control. These factors are usually determined by the methodology chosen for the
analysis. Under ideal conditions it is possible to achieve relative standard deviation
in concentrations as low as about 0.5 % which enables the determination of
microquantities of components. The precision of spectrophotometric method also
depends on concentration of the determinant. Visual methods generally give results
with a precision of 1 - 10 %. The precision of the photometric method is of course,
higher and varies from 0.5 - 2 % under suitable measuring conditions.
11
The precision attainable is a function of the absorbance measured. The error
observed is, as expected, very large on lower concentrations. When intensely
colored solutions are measured, only an insignificant part of the radiation is
transmitted and on the logarithmic absorbance scale the gradations are so close that
the reading error is very high. Precision is conveniently expressed in terms of the
average deviation from the mean or in terms of standard deviation. When applied to
small sets of data with which the analytical chemists work, the standard deviation is
the most reliable estimate of the indeterminate uncertainty. When the standard
deviation turns out to be approximately proportional to the amount present in the
formation on the precision can be expressed in percent by using the coefficient of
variation. Mathematical equation for the calculation of coefficient of variation is
given below
CV = (s × 100)/ x
where s = standard deviation and x = arithmetic mean of a series of measurements.
1. II. 8 DETECTION LIMIT
Detection limit is the smallest concentration of a solution of an element that
can be detected with 95 % certainty29,30. This is the quantity of the element that gives
a reading equal to twice the standard deviation of a series of ten determinations
taken with solutions of concentrations which are close to the level of the blank.
Several approaches for determining the detection limit are possible, depending on
whether the procedure is an instrumental or non-instrumental. Based on the standard
deviation of the reagent blank and the slope of the calibration curve of the analyte,
the detection limit (DL) may be expressed as,
DL = (3.3 σ)/ S
where σ = standard deviation of the reagent blank
S = slope of the calibration curve
The slope S may be estimated from calibration curve of the analyte. The estimate of
σ may be measured based on the standard deviation of the reagent blank.
1. II. 9 QUANTITATION LIMIT
The quantitation limit is generally determined by the analysis of samples
with known concentrations of analyte with those of blank samples and by
establishing the minimum level at which the analyte can be quantified with
12
acceptable accuracy and precision31,32. Based on the standard deviation of the reagent
blank samples and the slope of the calibration curve of the analyte, the quantitation
limit (QL) may be expressed as,
QL = (10 σ)/ S
where σ = standard deviation of the reagent blank
S = slope of the calibration curve
The slope S may be estimated from calibration curve of the analyte. The
estimate of σ may be measured based on the standard deviation of the reagent blank.
1. II. 10 COMPARISON OF THE RESULTS
The comparison of the values obtained from a set of results with either (i) the
true value or (ii) other sets of data makes it possible to determine whether the
analytical procedure has been accurate and / or precise, or if it is superior to another
method.
There are two common methods for comparing results33,34: Student’s t-test
and the variance ratio test (F-test).
These methods of test require knowledge of what is known as the number of
degrees of freedom. In statistical terms this is the number of independent values
necessary to determine the statistical quantity. Thus a sample of ‘n’ values has ‘n’
degrees of freedom, whilst ∑ ( xx )2 is considered to have n-1 degrees of freedom,
as for any defined value of x only n-1 values can be freely assigned, the nth being
automatically defined from other values.
(i) Student’s t-test
This is a test used to compare the mean from a sample with some standard
values and to express some level of confidence in the significance of the
comparison. It is also used to test the difference between the means of the two sets
of data x 1 and x 2.
t = [(x - µ)√n] / s
where s = standard deviation, x = arithmetic mean of a series of measurements, µ is
the true value and n is the number of trials of the measurements.
13
It is then related to a set of t-tables33,34 in which the probability of the t-value
falling within certain limits is expressed, either as a percentage or as a function of
unity relative to the number of degrees of freedom.
This method is also used to compare the values of the mean and precision of
the test method with those of the reference method. The value of ‘t’ when comparing
two sample means x 1 and x 2 is given by the expression,
t =(x1 -x2)
(1/n1) - (1/n2)Sp
where Sp is the pool standard deviation, calculated from two samples standard
deviations S1 and S2 as follows
Sp= (n1 - 1)S1
2 + (n2 - 1)S2
2
(n1 + n2 -2)
where n1 and n2 the number of trials of first and second method.
(ii) The Variance Ratio Test (F-test)
This is used to compare the precisions of two sets of data of two different
analytical methods or the results from two different laboratories. It is calculated
from the following equation34,35
F = SA2 / SB
2
The larger value of S is always taken in the numerator so that the value of
‘F’ is always greater than unity. The value obtained for F is then checked for its
significance against values in the F- table calculated from an F–distribution
corresponding to the numbers of degrees of freedom for the two sets of data.
1. II. 11 COLOR DEVELOPMENT
There are only a few elements, which give sufficient intense absorption by
themselves and are spectrophotometrically measurable. Majority of the substances
are generally determined indirectly in a variety of ways, such as
i. Substances may be converted by a suitable reagent to an absorbing product
ii. Adding complexing agent to get colored complexes and so on.
iii. Organic complexing agents are found to be more selective and sensitive
color developing agents.
14
1. II. 11. 1 Requirements of a Color Developer
A color developer should possess a high molar absorptivity, high selectivity
and the spectrum of the complex should be significantly different from that of the
reagent.
1. II. 11. 2 Criteria for Satisfactory Spectrophotometric Analysis
Eventhough spectrophotometric methods are versatile in nature, in order to
have successful and satisfactory result, the process of analysis need careful
operations. Since the color development in spectrophotometry involves diverse type
of reactions, a number of points need to be ensured before applying the method for a
particular application. Some of the points have to be considered are discussed in the
following sections.
1. II. 11. 3 Specificity of the Color
Very few reactions are specific for a particular substance, but may give
colors for a small group of related substances only and because of this it is important
to control the operational procedure so that the color is specific for the component
being determined. This may be achieved by isolating the substance by the normal
methods of analysis. But these separation methods are often tedious and time
consuming. Further there is every possibility of appreciable loss of the analyte
during these separations.
The specificity in colorimetric reactions can be achieved by introducing
other complex forming compounds. These are required to suppress the action of
interfering substance by the formation of complex ions or of non-selective
complexes. When the colorimetric reaction takes place within well-defined limits of
pH, adjustment will also sometimes help to achieve the desired specificity in certain
cases. The methods of selective absorption, chromatographic separations and ion
exchange separations are also of use in certain cases.
Solvent extraction method also finds its application in achieving specificity
in the spectrophotometric determinations. The interfering substances are removed by
extraction with an organic solvent, sometimes after suitable chemical treatment.
Alternatively the substance to be determined can also be isolated from the
15
interfering species by converting it into an organic complex, which is then
selectively extracted into a suitable organic solvent.
1. II. 11. 4 Proportionality Between Color and Concentration
For colorimeters, it is important that color intensity should increase linearly
with concentration of the compound to be determined. This is not necessary for
photoelectric colorimeters or spectrophotometers. Since a calibration curve may be
constructed relating the instrumental reading of the color with the concentration of
the solution. It is desirable that the system follows Beer’s law even when
photoelectric colorimeters are used.
1. II. 11. 5 Stability of the Color and Clarity of the Solutions
The color produced must be stable so as to allow accurate readings to be
taken. Stability of color is influenced by experimental conditions like temperature,
pH etc. The solution must be free from precipitate if comparison is to be made with
a clear standard. Turbidity scatters as well as absorbs the light.
1. II. 11. 6 Reproducibility and Sensitivity
The colorimetric procedure must give reproducible results under specific
experimental conditions. The reaction need not necessarily represent a
stoichiometrically quantitative chemical change. It is desirable, particularly when
minute amounts of substances are to be determined, that the color reactions be
highly sensitive. It is also desirable that the reaction product absorbs strongly in the
visible rather than in the ultraviolet region, as the interfering effect of other
substances is usually more pronounced in the ultraviolet region.
1. II. 12 CHOICE OF SOLVENT
The solvent which is to be used in colorimetric or spectrophotometric
determinations must meet certain requirements. It must be a good solvent for the
substance under determination. Before using a particular solvent, it must be ensured
that it does not interact with the solute. The solvent must not show significant
absorption at the wavelength to be employed in the determination.
For inorganic compounds, water normally meets these requirements, but for
majority of organic compounds, it is necessary to use an organic solvent. All
16
solvents show absorption at some point in the ultraviolet region and care must be
taken to choose a solvent for a particular determination which does not absorb in the
requisite wavelength region. Any impurities present in the solvents may affect the
absorption at certain wavelength and it is therefore, essential to employ materials of
the highest purity.
1. II. 13 LIMITATIONS
The most common and unrecognized problem in measuring the absorbance is
the stray light error. All wavelength isolation devices tend to produce some low
intensity radiations at wavelengths other than the desired one. This is usually due to
the optical imperfections, or simply from scattered light due to dust particles on
optical surface. Because one has usually selected a wavelength at which the
compound of interest absorbs most strongly, the stray light falling on the sample is
of wavelengths at which the compound does not absorb strongly. Thus the stray light
errors will result in a negative bias for absorbance readings which can be
represented in the equation.
Tobs = (Ttrue + ρ)/ (1+ρ)
where ρ is the fraction of all the light coming from the wavelength isolation
device, which is stray light and Tobs and Ttrue are the observed and true transmittance,
respectively. Normally the absolute amount of stray light tends to be relatively
constant with respect to the wavelength. But the fraction of stray light is highly
wavelength dependent because the amount of energy of the selected wavelength
depends on the source intensity at that wavelength. Thus, stray light errors are most
predominant at long and short wavelengths and when high absorbance is measured.
A common error encountered when making the measurements is called finite
slit width effect. The exit slit of the monochromator subtends a portion of the
dispersed continuum from the grating or prism. If any light is to pass through the slit
it must have a finite width. However, due to its width, more than one wavelength of
light, called the bandwidth, emerges. If the spectral band width is too wide, negative
deviation from Beer’s law occurs resulting in a false absorbance measurement.
17
Errors also occur when distilled water blank is used instead of a true blank
for 100 % transmittance or baseline reading. Eventhough there are no known
absorbing species in distilled water as well as in the blank reagent solution, the
difference in the refractive indices between the sample solution and the reference
solution must kept reasonably close. Even when the incident light is highly
collimated and falls on the cell window at normal incidence, a small fraction of the
light is reflected back at each interface where there is refractive index difference
because the sample and reference windows are of the same composition.
1. II. 14 APPLICATIONS
The greatest use of spectrophotometry lies in its application to quantitative
measurements. The reasons for this stem from the ease with which most
spectrophotometric measurements can be made, their sensitivity and precision and
the relatively low cost of instrumental purchase and operation. A variety of
techniques have been developed for different types of samples. Direct
determinations are made when the analyte molecule contains a chromophore, thus
allowing the direct measurement of its absorbance. Standards must be used to
determine the absorptivity so that concentration can be calculated by using the
equations or by establishing a calibration plot from which the concentration can be
determined graphic interpretation or by regression analysis. Indirect determinations
are commonly used when the analyte molecule does not contain a suitable
chromophore. In these instance the analyte is made to quantitatively react with
molecules containing a chromophore and correlating the diminution of absorbance
with the concentration of analyte or by reacting with a reagent, which produces a
chromophoric groups.
Spectrophotometric analysis continues to be one of the most widely used
analytical technique available. Many methods are available for a variety of analytes
(such as colored, colorless, natural, synthetic, inorganic and organic analytes) and
sample types ranging from in-situ biological assays to the determination of trace
elements in steels. Many medical diagnostic test kits are used in photometric
measurements. Diabetics commonly use blood-glucose analysis kits based on the
glucose oxidase enzyme reaction that secondarily produces a colored product. In
food industry, winemakers have long recognized the effect of iron levels on the taste
18
of wines and consequently are one of the largest users of 1, 10-phenthroline for
determining iron spectrophotometrically. A common field test for chlorine in
swimming pools and drinking water is based on the color produced by the action of
chlorine on o-tolidine.
Many compilations of methodology for a variety of analytes and sample
types that are regularly updated are available35-37. Other general sources for
spectrophotometric analysis are commonly consulted and found helpful38-40.
Standard methods specific to certain industries and areas of study are very useful
sources when specific sample types are being considered, such as water, waste water
and pharmaceuticals41,42.
19
PART III
INTRODUCTION TO SINGLE CRYSTAL X-RAYDIFFRACTION
1. III. 1 X-RAY CRYSTALLOGRAPHY
1. III. 2 DIFFRACTION OF X-RAYS BY CRYSTALS
1.III. 3 EXPERIMENTAL
1. III. 4 COMPUTATIONS
1. III. 5 PRESENT INVESTIGATIONS
1. III. 6 REFERENCES
20
1. III. 1 X-RAY CRYSTALLOGRAPHY
The study of the theory of crystallography has caught the attention of many
scientists since long time. The first definite contribution was from Kepler in the year
1611. Stensen pointed out the existence of the characteristic interfacial angles in the
crystals which later lead to the Miller indexing of the planes and subsequently the
classification of crystals under 32 classes independently by Hessel and Godolin.
Neumann showed that the laws of symmetry that hold for the external faces hold
good even for the properties exhibited by the crystals. The contribution of Huygens
and Hauy considered the building blocks of the crystals. Based on the atomic theory
of Seeber, Bravais proved the existence of 14 lattices which are currently named
after him. Later investigations using group theoretical approach by Fedorov and
Schoenflies proved the presence of 230 space groups. Max von Laue worked out the
theory of gratings with double periodicity as present in crossed gratings. As the
science of optics extended beyond the visible spectrum into the domain of very short
waves like X-rays, which were discovered by Rontegen, the fine structure of crystals
was demonstrated by Friedrich and Knipping in 1912. This was the starting point of
X-ray Crystallography which saw a rapid development of theoretical ideas and
techniques to unravel the structures of the wide range of crystalline substances, both
natural and synthetic.
1. III. 2 DIFFRACTION OF X-RAYS BY CRYSTALS
Crystal is a homogeneous solid43,44 and is defined as three dimensional
periodic arrangement of atoms or molecules. Within a crystal, the atoms or
molecules are arranged in an orderly manner. Such periodically repeating motif in
three-dimensions forms a natural grating for diffraction of waves having suitable
wavelength. Max von Laue proposed that X-rays are electromagnetic waves and
these are diffracted by a crystal45. Later, Friedrich and Knipping46 showed the
diffraction of X-rays from single crystals of copper sulphate pentahydrate.
The periodic repetitions of motif or basis are represented by three shortest
non-coplanar vectors43, 44, 47, 48 a, b and c and are called primitive vectors. The
parallelopiped generated by these vectors is called a unit cell or primitive cell. All
possible linear combinations of these three unit vectors generate an infinite array of
21
discrete points in space referred to as lattice of the crystal. The position of a lattice
point is represented by a vector,
r
=lambnc
where l, m and n are integers. Later, W.L. Bragg and W.H. Bragg treating
diffraction as reflection from planes in the lattice, deduced the simple equation,
2dhklsin = n
known as Bragg’s law, where is the glancing angle for incident X-rays of
wavelength , dhkl is the interplanar spacing of planes characterized by the Miller
indices hkl and n is order of diffraction.
1. III. 3 EXPERIMENTAL
The process of crystallization is the ordering of randomly arranged ions,
atoms, or molecules to take up regular positions and shape in the solid state. It
involves the phenomenon of nucleation and it may be considered to be in dynamic
equilibrium between particles in the fluid phase and solid phase from saturated
solutions. Several techniques are available for crystallization of small molecules
such as slow evaporation, slow cooling, diffusion methods etc.
Single crystals of all the compounds presented in this thesis are obtained
from slow evaporation technique. Crystals are examined under a polarizing
microscope before mounting in the goniometer head for data collection.
1. III. 4 COMPUTATIONS
Data collection: X-AREA; CrysAlis CCD (Oxford Diffraction, 2004);
SMART (Bruker, 1998); COLLECT; cell refinement: X-AREA; CrysAlis RED
(Oxford Diffraction, 2004); SAINT –Plus; DENZO ; data reduction: X-AREA;
CrysAlis RED; SAINT -Plus; DENZO, SCALEPACK and COLLECT; program(s)
used to solve structure: SHELXS97; SHELXTL/PC; SHELX97; SHELXS97;
program(s) used to refine structure: SHELXL97; SHELXTL/PC; molecular graphics:
XP in SHELXTL-Plus; SHELXTL/PC) and MERCURY; ORTEP-3 and PLATON;
software used to prepare material for publication: SHELXL97; PLATON 49-57.
22
1. III. 5 PRESENT INVESTIGATIONS
The work described in chapters 2-5 deals with the spectrophotometric
determination of some antimalarial, antiarthritis, antihypertensive and anti
reteroviral drugs. Last chapter is devoted for the synthesis and characterization some
chalcones and their derivatives.
The reagents and drugs used in this study are given below:
N
N+
CH3
NH2
CH3
NH2
Safranin O
N
H3CO
H
NH2
Variamine blue
NH
NH
O
O
NaO3S
SO3Na
Indigo CarmineNH
CH3
CH3 CH3
N
CH3
SO3-
SO3-
Na+
+H
Xylene Cyanol FF
NNCH3
CH3 CH3
CH3
N+ CH3CH3
Crystal Violet
O
O
O . H2O
Ninhydrin
CH3
SN
- Na
+
OO
Cl
Chloramine-T
O
O
O
CH3
CH3
CH3
OO
H
Artemisinin
23
CH3 – CHO
Acetaldehyde
Na2[Fe(CN)5NO]
Sodium nitroprusside
O
OH
O
CH3
CH3
CH3
OO
H
Dihydroartemisinin
O
O
O
CH3
CH3
CH3
OO
H
O
OH
O
Artesunate
O
O
O
CH3
CH3
CH3
OO
H
CH3
Beta – arteether
O
O
O
CH3
CH3
CH3
OO
H
CH3
Beta - artemether
O
OHH
HH
OHOH
H NH3. Cl-
H
OH
+
Glucosamine Hydrochloride
O
OHH
HH
OHOH
H NH3
H
OH
+
2
. SO42-
Glucosamine Sulphate
N
O
H
N
O
OH
H
H3CO
Carvedilol
NH
NNN
OCH3
Nevirapine
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