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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