CHAPTER 5

EXPERIMENTAL METHODS

CHAPTER OVERVIEW

Section 5.1 gives an introduction of the experimental methods.

The importance of the non-destructive method is explained. Section

5.2 presents the physical parameters to be determined and

introduces various apparatus used in the work. Section 5.3 describes

the working of a refractometer and explains how the refractive index

of a liquid is measured accurately and precisely with Abbe'

refractometer. Section 5.4 describes various parts of ultrasonic

interferometer and explains how it is used to determine ultrasonic

velocity accurately. Section 5.5 describes the working of UV-Visible

Spectrophotometer In general and explains how Perkin Elmer

Spectrophotometer IS used for the determination of UV-Visible

absorption wavelengths. Section 5.6 describes the determination of

density of a liquid using Pyknometer and electronic weighing

balance.

5.1 INTRODUCTION

Experiments are the part and parcel of all branches of Science.

The present chapter gives an idea of the instruments used and how

various experiments are conducted with them. Sources of errors and

precautions are also discussed wherever necessary.

Physical and chemical methods are the two ways by which the

structural changes can be assessed. In the present work, physical

methods are used for the determination of the photochemical change

of certain medicines due to solar exposure. The physical parameters

used are density, ultrasonic velocity, refractive index and UV-Visible

absorption wavelengths along with the corresponding absorbance.

The physical method has got an advantage that the molecules of the

73

substances under study remain unaltered during the experiment. In

short, physical method is a non-destructive one. Non-destructive

methods are superior to other destructive chemical methods. [Walter,

1958]

5.2 EXPERIMENTAL SET UP IN THE PRESENT STUDY

The focus of the present work is the determination of structural

changes of certain commonly used medicines due to solar exposure.

This is a potent area of research now. Literature survey reveals that a

lot of works are being carried out all over the world in this regard. In

the present work, acoustic, optic and opto-acoustic parameters viz.

specific optic impedance (Zo), specific optical volume (v), specific

opto-acoustic velocity (TJ), adiabatic compressibility C{is) , Rao's

specific sound velocity (r) and specific acoustic impedance (ZA) have

been used. Since the above mentioned derived parameters are

relations connecting sound velocity 'U, density p and index of

refraction n, the experiment is focused on the accurate determination

of these physical parameters at different temperatures. Fig.5.1 gives

the experimental set up for the present study.

The ultrasonic velocities at different temperatures were

determined with the help of a single crystal ultrasonic interferometer

(MITTAL Enterprises-Model No.F-81) at a frequency of 2 MHz having

an accuracy of ± 0.1 mj s. The refractive index measurements were

done using Abbe Research refractometer (ADVANCE model No.R-8)

having least count 0.0001. For the determination of densities, a

12cm3 double stem pyknometer and an electronic balance (ACCULAB

model No.ALC-210.4) were used. The least count of the balance

is O.lmg. The absorption spectra were taken uSIng a computerized

UVjVisible spectrophotometer manufactured by Perkin-Elmer Ltd,

USA with trade name 'UV win Lab, Lambda 25 UV jVIS'

74

\I~

Fig.5.t Experimental set up

The temperature was kept constant using a thermostatically

controlled water circulating arrangement with an accuracy of ± 0.1 K.

All the measurements were performed at five different temperatures

viz. 298, 303, 308, 313 and 318 K. Each observation was repeated

twenty five times and the results were averaged.

A detailed description of various instruments along with their

background theory is given in the next sections.

5.3 REFRACTIVE INDEX MEASUREMENT

Like density, surface tension and viscosity, refractive index is a

very useful physical property of liquids. Being an easy and non­

destructive method, refractive index is used for the identification of

liquids. For a large group of medicines, refractive index is used for

the assessment of purity (quality control). Refractometer is an

instrument used for the determination of refractive index. [Donald et

aI., 1976]

75

5.3.1 WORKING PRINCIPLE OF A REFRACTOMETER

The speed of light in vacuum is always the same, but when

light moves through any other medium it travels more slowly since it

is constantly being absorbed and re-emitted by the atoms in the

material. The ratio of the speed of light in vacuum to the speed of

light in another substance is defined as the index of refraction

(refractive index or n) for the substance. Whenever light changes

speed as it crosses a boundary from one medium into another its

direction of travel also changes as in fig.5.2. In other words, it is

refracted. (In the special case of the light traveling perpendicular to

the boundary there is no change in direction upon entering the new

medium.) The relationship between the speed of light in the two

media (VA and V8), the angles of incidence eA and refraction 88 and the

refractive indices of the two media (nA and n8) is shown in equation

number 5.1.

Light moving at speed "A

Medium A Refractive Index "A

.. ,_.....

,e. , B

Light moving at speed v8

Fig.5.2 Refraction of light. The speed of light in medium A is greater

than the speed of light in medium B

76

VA sin8A n8 -=--=- (5.1)

Thus, it is not necessary to measure the speed of light in a

sample in order to determine its index of refraction. Instead, by

measuring the angle of refraction, and knowing the index of

refraction of the layer that is in contact with the sample, it 1s

possible to determine the refractive index of the sample quite

accurately. Nearly all refractometers utilize this principle, but may

differ in their optical design.

5.3.2 THE ABBE REFRACTOMETER

Illuminating Prism

Sample

Refracting Prism

Light Dark

Fig.5.3 Cross section of part of the optical path of an Abbe

refractometer.

In the Abbe refractometer the liquid sample is sandwiched into

a thin layer between an illuminating prism and a refracting prism as

shown in fig.5.3. The refracting prism is made of a glass with a high

refractive index (e.g. 1. 75) and the refractometer is designed to be

used with samples having a refractive index smaller than that of the

77

refracting prism. A light source is projected through the illuminating

prism, the bottom surface of which is ground (i.e., roughened like a

ground-glass joint), so each point on this surface can be thought of

as generating light rays travelling in all directions. Inspection of

fig.5.3 shows that light travelling from point A to point B will have

the largest angle of incidence ei and hence the largest possible angle

of refraction er for that sample. All other rays of light entering the

refracting prism will have smaller er and hence lie to the left of

point C. Thus, a detector placed on the back side of the refracting

prism would show a light region to the left and a dark region to the

right.

Prism lock

S I Amici prismca e

d.

lamp a Justment

Lamp

Eye piece Thermometer

Scale adjustment

Fig.5.4 Abbe Refractometer - side views and front view

Samples with different refractive indices will produce different

angles of refraction (see equation 5.1 above and recall that the angle

of incidence and the refractive index of the prism are fixed) and this

will be reflected in a change in the position of the borderline between

the light and the dark regions. By appropriately calibrating the scale,

78

the position of the borderline can be used to determine the refractive

index of any sample. This is the essential principle of Abbe

refractometer. The Abbe refractometer is shown in fig.5A.

5.3.3 NECESSITY OF A MONOCHROMATIC SOURCE

In most liquids and solids the speed of light, and hence the

index of refraction, varies significantly with wavelength. Thus, for the

most accurate measurements it is necessary to use monochromatic

light. The most widely used wavelength of light for refractometry is

the sodium D line at 589 nm.

If white light were used In the simple Abbe' refractometer

shown in fig.5.3, dispersion would result in the light and dark

borderline being in different places for different wavelengths of light.

The resulting 'fuzziness' of the borderline would make precise work

impossible. However, many Abbe' refractometers are able to operate

satisfactorily with white light by introducing a set of 'compensating

prisms' into the optical path after the refracting prism. These

compensating prisms are designed so that they can be adjusted to

correct (i.e., compensate for) the dispersion of the sample in such a

way that they reproduce the refractive index that would be obtained

with monochromatic light of 589 nm, the sodium D line.

As mentioned earlier, the speed of light in a substance v IS

lower than that in vacuum c since the light is being absorbed and

reemitted by the atoms in the sample. Since the density of a liquid

usually decreases with temperature, it is not surprising that the

speed of light in a liquid will normally increase as the temperature

increases. Thus, the index of refraction normally decreases as the

temperature increases for a liquid. For many organic liquids the

index of refraction decreases by approximately 0.0005 for every 1Deincrease in temperature. However, for water the variation is only

about -0.000 1re.

79

Many refractometers are equipped with a thermometer and a

means of circulating water through the refractometer to maintain a

given temperature. Most of the refractive index measurements

reported in the literature are determined at 20 or 25°C.

5.3.4 ADVANTAGES

Abbe' refractometers have the following advantages

1. A white-light source may be used, but the reading will be

that for the sodium D line.

2. Only a few drops of the liquid are required.

3. Allows for temperature control of prisms and sample.

4. Compensating Amici prisms allow one to compensate for

the wavelength dependence of the refractive index.

5.3.5 PROCEDURE

1. To introduce the sample unlock the prism, lift the top

pnsm, spread a few drops of the sample on the bottom

prism, close the prisms slowly, and lock the prisms again

as in fig.5.5.

/'

Fig.5.5 Placing of sample in the refractometer prism

2. Turn the instrument on (on/off switch).

3. Focus the eyepiece on the scale by rotating it.

4. Turn the scale adjustment so that the critical ray

boundary is visible in the top part of the viewer (a dividing

line between light and dark halves is visible).

80

5. Turn the Amici prism adjustment so as to achromatize the

boundary. Different cases are shown in fig.5.6. The centre

image shows proper achromatization (white color - sharp

boundary).

Fig.5.6 Different cases of critical ray bO!Jndary.

6. Turn the scale adjustment so that the boundary between

light and dark coincides with the center of the cross hairs

as shown in fig.5.7.

Fig.5.7 The correct scale adjustment

7. Read and record the refractive index on the top scale in the

lower part of the viewer (the bottom scale is for the

concentration of sugar in water and can be ignored). Three

81

decimal places can be read, the fourth place is estimated

as in fig.5.8.

1.34 1.35

Fig.5.B Taking of readings

8. The image in fig.5.8 shows a reading of 1.3433 (notice the

smallest division is 0.0005).

9. If the specific dispersion is required, read and record the

amici prism adjustment knob.

10. Read and record the temperature on the thermometer.

11. Keep the prisms clean (top and bottom) as shown in

fig.5.9. Use water to remove water soluble compounds,

toluene or petroleum ether for water insoluble compounds.

Be sure not to scratch the prisms.

12. Leave the prisms in an open position so that they can be

air dry.

Fig.5.9 Cleaning the prisms

82

5.3.6 SOURCES OF ERROR AND PRECAUTIONS

A typical laboratory refractometer can determine the refractive

index of a sample to a precision of± 0.0001. However, small amounts

of impurities can cause significant changes in the refractive index of

a substance. Thus the compound should be rigorously purified.

Another possible source of error is miscalibration of the

refractometer. This is readily checked by using a sample of known

refractive index. Distilled water is a particularly convenient standard

since it is nontoxic, readily available in pure form, and its refractive

index varies only slightly with temperature as shown in table 5.1. If

you find that the index of refraction of the standard is consistently

off by more than 0.0005 from the expected value, the refractometer is

to be calibrated .

. Substance __ J n15 _L n'f,0 l n'f,5 j

I lsopropanol __

· _

ll.380� 1.3772 1.3749

I i

I

I Acetone 1.3616 1.3588 1.3560

________ _J I

i Ethyl Acetate 1.3747 1.3742 1.3700

I I i ·--

I Water 1.3334 1.3330 1.3325

L-··--········-·············-······ ............................................. ··-···"·· ········-

Table 5.1 Temperature dependence of refractive index for selected

substances.

83

Probably the most common source of error In analog

refractometer is misreading of the scale. If the index of refraction

determined seems inconsistent with other data, the measurement

may be repeated. Since the index of refraction depends on both the

temperature of the sample and the wavelength of light used, these

are both indicated when reporting the refractive index.

n'b° = 1.3742

In the example gIven above, the italicized n denotes refractive

index, the superscript indicates the temperature in degrees Celsius,

and the subscript denotes the wavelength of light (in this case the D

indicates the sodium D line at 589 nm).

5.3.7 STRUCTURAL INFORMATION

The refractive index does not provide detailed information

about a molecule's structure, and it is not usually used for this

purpose since spectroscopic techniques are much more powerful at

revealing details of molecular structure. One structural factor that

influences the refractive index of a sample is its polarizability.

Substances containing more polarizable ("soft") groups (e.g., iodine

atoms or aromatic rings) will normally have higher refractive indices

than substances containing less polarizable ("hard") groups (e.g.,

oxygen atoms or alkyl groups). See table 5.2.

Substance 2-lodoethanol 2-Fluoroethanol Benzene Cyclohexane

n~O

I

1.5720 1.3670 1.5010 1.4260

i

Table 5. 2 Effect of polarizable groups on refractive index.

84

5.4 DETERMINATION OF ULTRASONIC VELOCITY

Refractive index is a parameter depending on the velocity of

light in the material. Like refractive index, ultrasonic velocity is a

characteristic property so that it can also be used for the

characterization of materials. The device used for the determination

of ultrasonic velocity of liquids is known as ultrasonic interferometer.

In such devices, ultrasonic waves are usually produced by a quartz

crystal.

5.4.1 ULTRASONIC INTERFEROMETER FOR LIQUIDS

Fig.S.lO Ultrasonic interferometer

An ultrasonic interferometer is a simple and direct device to

determine the ultrasonic velocity in liquids with a high degree of

accuracy. It is shown in fig.S.IO. With the help of this apparatus

several PhD thesis bagged honours and innumerable research papers

are published in national & international journals. Velocity

8S

measurement combining with other physical quantities provides

information of more than 30 Parameters.

5.4.2 PARTS OF ULTRASONIC INTERFEROMETER

The parts of an Ultrasonic interferometer are shown in fig.5.ll.

~ THE MEASURING CELL.

It is a specially designed double walled cell for taking the

experimental liquid. Temperature can be kept constant by circulating

water. A quartz crystal is arranged at the bottom of the cell. There is

provision for giving HF input to the crystal. A polished metal reflector

is arranged at the top of the measuring cell. The position of the

reflector can be adjusted in the liquid with the help of a micrometer

screw.

-~-- Micrometer

R.F Input --...~.. t:i1

1'I2=.ti1~- Reflector

:++-- Experimental liquid

~=~::::--- Quaryz Crystal

H--Screw

The measuring cell

Fig.5.t1 Parts of Ultrasonic interferometer

86

� THE HIGH FREQUENCY GENERATOR

The 2 MHz electronic oscillator together with the quartz crystal

having a natural frequency of 2 MHz arranged at the bottom of the

measuring cell form the generator of ultrasonic waves. The quartz

crystal acts as a transducer. A micro ammeter is used to observe the

changes of current during the variation of the position of the

reflector. There is one control knob for adjusting the gain of the

output and another for the zero setting of the micro ammeter.

5.4.3 ADJUSTMENT OF ULTRASONIC INTERFEROMETER

The correctness of the measurement depends on the systematic

and careful observation. To begin with the following adjustments are

to be done.

1. Insert the cell in the square base socket and clamp to it

with help of a screw provided on one of its sides

2. Unscrew the knurled cap of the cell, lift it away, take the

experimental liquid inside the lower cylindrical portion and

screw the knurled cap back.

3. Circulate water through the chutes in the double walled

cell and maintain the desired temperature.

4. Connect the HF generator with cell by a coaxial cable

provided with the instrument .

. 5. The distance between the quartz crystal and the reflector is

initially kept at minimum and is increased gradually by

turning the micrometer screw. The periodic deflection of

the micro ammeter is observed. The knob labeled 'Gain' is

adjusted conveniently so that the deflection is not either

too small or out of scale. The knob labeled 'Adj' is adjusted

so that the 'minimum' position of the needle of the micro

ammeter coincides with the zero of the ·scale.

87

5.4.4 WORKING PRINCIPLE OF AN ULTRASONIC INTERFEROMETER

The principle used in the measurement of velocity U is based

on the accurate determination of the wavelength in the medium.

Ultrasonic waves of known frequency (f = 2MHz) are produced

by a quartz crystal fixed at the bottom of the ultrasonic cell. These

waves are reflected by a movable metallic plate kept parallel to the

quartz crystal and standing waves are formed in the medium as

shown in fig. 5.12. This acoustic resonance gives rise to an electrical

reaction on the generator driving the quartz crystal.

If the distance between crystal and plate is now increased or

decreased and the variation is exactly one half of a wave length or

multiple of it, anode current becomes maximum or minimum as in

fig. 5.13. From the knowledge of wavelength the velocity U can be

obtained by equation 5.2

Velocity = Wavelength x Frequency 5.2

Metal reflector

Quartz crystal

Fig.5.12 Standing waves in the liquid

88

If the variation in the velocity with temperature is to be

studied, water at various desired constant temperatures is made to

circulate through the double walled jacket of the cell.

Crystal

current

Position of reflector

Fig.5.13 Position of reflector Vs crystal current.

5.4.5 MEASUREMENT OF ULTRASONIC VELOCITY

The measuring cell is connected to the output terminal of the

high frequency generator of 2MHz through a shield cable. The cell is

filled with 12 ml of the experimental liquid. The adjustments are

done as mentioned earlier. The micrometer screw attached to the

reflector is moved slowly till the micro ammeter shows a maximum.

Reading of the micrometer screw is noted as x1. The micrometer

screw is then turned slowly and carefully so that the current

decreases to minimum and then increases to maximum n times. The

final reading of the micrometer screw is noted as x2• The distance

between the 1st maximum and nth maximum,

(5.3)

89

If ii. is the wavelength of light used,

ii. d =n-

2 (5.4)

Once the wavelength ii. is known, the velocity U of the liquid can

be calculated using the equation

2d

u = fil = f-n

Where, f is the constant frequency of the interferometer (2 MHz).

(5.5)

The experiment is repeated at different temperatures with the

help of thermostatically controlled water circulating arrangement

having an accuracy of± 0.1 K.

5.4.6 PARAMETERS RELATED TO ULTRASONIC VELOCITY

1. Compressibility

2. Effective Debye Temperature

3. Excess Enthalpy

4. Hydrogen Bonding

5. Intermolecular Free Length

6. Solvation Number/ Hydration Number

7. Vander Wall's Constant

8. Rao's Constant

9. Wada Constant

10. Molecular Interaction

11. Proton Relaxation Rate

12. Relative Association

13. Relaxation Time and Relaxation Strength

14. Acoustic Impedance

90

5.5 UV-VISIBLE ABSORPTION SPECTRUM

Different substances absorb different wavelengths of light and

this can be used to identify the substance, the presence of particular

metal ions or particular functional groups in organic compounds.

The amount of absorption is also dependent on the concentration of

the substance if it is in solution. Measurement of the amount of

absorption can be used to find concentrations of very dilute

solutions. An absorption spectrometer measures the way that the

light absorbed by a compound varies across the UV and visible

spectrum. Some colourless substances also absorb light - but in the

ultra-violet region. Since we can't see UV light, we don't notice this

absorption.

5.5.1 A SIMPLE DOUBLE BEAM SPECTROMETER

The full diagram of a UV-Visible Spectrometer IS shown in

fig.5.14. The various parts and the working of each stage are given

below.

a Slit

light source

Referencecell

,.--..,..-----, Detectorand

'---r---' computer

Chart recorder

Fig.5.14 double beam UV-Visible Spectrometer

91

5.5.2 PARTS OF A DOUBLE BEAM UV-VISIBLE SPECTROMETER

It is a very sensitive instrument. The output IS usually

analysed using a computer. The reliability of the instrument is high.

The list of absorption peaks as well as the complete spectrum

connecting absorbance against wavelength can be seen on the

monitor itself. The wavelength range for taking the spectrum can be

given on the monitor. A double beam UV-Visible spectrometer shown

in fig.5.14 has the following parts.

1. The light source

2. The diffraction grating and the slit

3. The rotating discs

4. The sample and reference cells

5. Thede~c~randcompu~r

6. The chart recorder

5.5.3 THE LIGHT SOURCE

It consists of a light source which gIves the entire visible

spectrum plus the near ultra-violet, covering the range from about

200 nm to about 800 nm. (This extends slightly into the near infra­

red as well). It is impossible to get this range of wavelengths from a

single lamp so that a combination of two is used - a deuterium lamp

for the UV part of the spectrum, and a tungsten/halogen lamp for

the visible part. A deuterium lamp contains deuterium gas under low

pressure subjected to a high voltage. It produces a continuous

spectrum in the part of the UV spectrum we are interested in. The

combined output of these two bulbs is focused on to a diffraction

grating.

5.5.4 THE DIFFRACTION GRATING AND THE SLIT

A diffraction grating splits light into its component colours

more efficiently than a prism. The blue arrows show the way the

92

various wavelengths of the light are spread to different directions.

The slit only allows light with a very narrow range of wavelengths

into the rest of the spectrometer. By gradually rotating the diffraction

grating, we can allow light from the whole spectrum (a tiny part of

the range at a time) into the rest of the instrument.

5.5.5 THE ROTATING DISCS

This is the most important part of a spectrometer. Each disc is

made up of a number of different segments. We are referring to those

machines which have three different sections - other designs may

have a different number.

The light coming from the diffraction grating and slit will hit

the rotating disc and one of three things can happen.

1. If it hits the transparent section, it will go straight through

and pass through the cell containing the sample. It is then

bounced by a mirror onto a second rotating disc. This disc

is rotating such that when the light arrives from the first

disc, it meets the mirrored section of the second disc. That

bounces it onto the detector. It is following the red path in

the diagram.

2. If the original beam of light from the slit hits the mirrored

section of the first rotating disc, it is bounced down along

the green path. After the mirror, it passes through a

reference cell. Finally the light gets to the second disc

which is rotating in such a way that it meets the

transparent section. It goes straight through to the

detector.

3. If the light meets the first disc at the black section, it is

blocked - and for a very short while no light passes

through the spectrometer. This just allows the computer to

make allowance for any current generated by the detector

in the absence of any light.

93

5.5.6 THE SAMPLE AND REFERENCE CELLS

These are small rectangular quartz containers. They are often

designed so that the light beam travels a distance of 1cm through the

contents. The sample cell contains a solution of the substance we are

testing - usually very dilute. The solvent is chosen so that it doesn't

absorb any significant amount of light in the wavelength range we

are interested in (200 - 800 nm). The reference cell contains just the

pure solvent.

5.5.7 THE DETECTOR AND COMPUTER

The detector converts the incoming light into a current. The

higher the current, the greater is the intensity of the light. For each

wavelength of light through the spectrometer, the intensity of the

light passing through the reference cell is measured. This is usually

referred to as 10 , The intensity of the light passing through the

sample cell is also measured for that wavelength - given the symbol,

1. If 1<10 , then obviously the sample has absorbed some of the light.

A simple bit of calculation is then done in the computer to convert

this into something called the absorbance of the sample - given the

symbol, A. The relationship between A and the two intensities is

given by

(5.6)

In most of the diagrams we come across, the absorbance

ranges from 0 to 1, but it can go higher than that. An absorbance

of 0 at some wavelength means that no light of that particular

wavelength has been absorbed. The intensities of the sample and

reference beam are both the same, so the ratio loll is 1. [0910(1) is

equal to zero. An absorbance of 1 happens when 90% of the light at

that wavelength has been absorbed - which means that the intensity

is 10% of what it would otherwise be.

94

In that case, I0/I is 100/IO (=10) and log10 (10) is 1.

An absorbance scale often runs from O to 1, but could go

higher than that in extreme cases where more than 90% of a

wavelength of light is absorbed.

5.5.8 THE CHART RECORDER

0.6

C l'O -E! 0.3

O..J.-r....,.......,;;..,r-r-,,-,-,-,-.,...,....,...,..-r-r...,...,,...,...,

200 300 400 500 600

Wavelength (nm)

Fig.5.15 A plot of absorbance against wavelength

Chart recorders usually plot absorbance against wavelength.

The output might look like fig.5.15. This particular substance has

what are known as absorbance peaks at 255 and 395 nm. How these

arise and how they are interpreted is discussed on another page.

5.5.9 DETERMINATION OF UV-VISIBLE ABSORPTION SPECTRUM

A computerized UV /Visible spectrophotometer manufactured

by Perkin-Elmer Ltd, USA with trade name 'UV Win Lab, Lambda 25

UV /VIS' is used for taking the absorption spectra of the liquid

medicines. This is shown in fig.5.16. The Lambda 25 has true

95

double-beam operation that provides the best possible stability and

allows references to be measured and corrected in real time.

However, the bandwidth of the Lambda 25 is fixed at 1 nm.

Applications include quantification of nucleic acids, proteins,

monitoring enzyme reactions, materials characterization,

pharmaceutical quality control, and colour control. UV/Visible

spectroscopy has become more sophisticated, providing higher

quality results faster and easier than ever. The details are as follows.

96

lIB

Fig.5.16 Perkin-Elmer - UV win Lab, Lambda 25 UV-Visible Spectro­

photometer.

1. Computerized UV-Visible Spectrophotometer

2. De ionized water

3. A 500 ml volumetric flask

4. A 5 ml pipette

5. 2 quartz cells

5.5.11 SETTING UP THE EXPERIMENT

The following procedure details how to get the instrument and

software up and running:

5.5.10 MATERIALS REQUIRED

1. Make sure that the beam path through the sample

compartment of the instrument is clear. The reference and

sample cell holders must be empty, or any accessory must

be properly installed, otherwise the instrument will not

initialize correctly.

2. Switch on the instrument and leave it for approximately 10

minutes to allow the lamp to warm up and stabilize.

3. Switch on the PC.

4. From the Windows Start menu, open the PerkinElmer

Applications group under Programs and start UV Win Lab,

which will have the name of the instrument set up during

the installation procedure, for example Lambda 800. The

UV WinLab software starts and the Methods window is

displayed.

5.5.12 ADJUSTMENTS OF THE SPECTROPHOTOMETER

When UV Win Lab was started, the Methods window was

displayed. This window has four tabs at the bottom which relate to

the four different types of method available. Select the menu 'Scan',

which is used for scanning spectra. Perform the operations as listed

below.

1. Click 'graph' menu. The Method Editor window 1s

displayed, with the first Scan method from the Methods

window loaded. Beneath the Method Editor window are

minimized windows for 'Data Region', 'Result Window' and

'Graph 1 as explained below'.

a) 'Data Region' is the temporary memory where data 1s

stored as long as the UV Win Lab software is running.

b) 'Result Window' is where the numerical results of the

current method or command are displayed.

c) 'Graph 1' is where the graphical results of the current

method or command are displayed

97

2. Select the 'Sample tab' by clicking on it.

3. Change the Sample Identity from the default name 'first

sample' to the actual name of the experimental liquid, the

method is now complete, but must be saved.

4. Select 'Save As' from the File menu. A file selector 1s

displayed.

5. Enter 'Scantest' as the Filename.

6. Select 'Auto zero on start'. Whenever a method is started,

the instrument will auto zero (perform background

correction) before the measurement is made.

7. Select 'Use next auto file name'. Future samples will be

automatically saved with the filename plus three digits

that are automatically updated for each sample.

5.5.13 EXPERIMENTAL PROCEDURE

1. Place a quartz cell containing deionized water in the

reference cell holder and close the cover. The deionized

water is a reference solution for the sample as it is the

solvent used to dilute the sample.

2. Click 'Start'. The instrument auto zeros and a blank

sample is requested.

3. Place the other quartz cell, again containing just deionized

water, in the sample cell holder and close the cover. This is

the blank solution. That is a solution without any of the

chemical you want to analyze.

4. Click 'OK'. The background correction is performed (which

takes a while) and then the sample is requested.

5. Empty the sample cell and re-fill it with the solution of the

experimental liquid made earlier.

6. Place the sample cell back in the holder and close the

cover.

7. Click 'OK'. The analysis is performed. During the analysis

the current readings are shown on the live display near the

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bottom of the UV WinLab window. When the scan is

complete the spectrum is displayed in the Graph 1 window.

5.5.14 VIEWING STATUS INFORMATION

1. Click the button, next to the name of the spectrum. The

status information for the spectrum is displayed in a new

window.

2. Click 'Instrument'. The instrument parameters are

displayed.

3. Click 'OK'. The Instrument window closes.

4. Click 'Close'. The Status Information window closes.

5. Select 'Print' from the 'File' menu. Graph 1 is printed.

6. Select 'Save' from the 'File' menu. A file selector is

displayed.

7. Enter the name of the experimental liquid as the 'Filename'

and click 'OK'. The spectrum is saved.

5.6 DENSITY MEASUREMENT USING PYKNOMETER

The density of liquids is accurately determined using a

pyknometer in conjunction with an electronic balance. The name

pyknometer is from the Greek word 'puknos' meaning density. It is a

tube like container and its pipette like part has a mark to show how

far to fill it and is bend into a U shape to facilitate immersion in a

temperature bath. Such a pyknometer consists of a cylindrical glass

bulb, to the ends of which pieces of glass tubes of narrow bore are

attached.

At the time of using pyknometer, the density of the liquid

1s determined by measurement of the weight of liquid occupying a

known volume. Before each weighing the volume of liquid is adjusted

so that it fills the vessel from the mark on one arm to the drawn out

tip of the other. The density varies considerably with temperature

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and hence every filling must be carried out In a thermostat at the

required temperature.

5.6.1 DETERMINATION OF DENSITY

To begin with, the pyknometer IS dried carefully and

weighed, then filled with distilled water and place in a thermostat for

ten or fifteen minutes. With the pyknometer the quantity of the liquid

is then adjusted so that liquid meniscus is at the mark on the

horizontal capillary. If the pyknometer contains too much liquid, the

excess may be removed by touching the constricted tip with a scrap

of filter paper. If it contains too little, catch a drop of liquid on the

end of a stirring rod and bring it in contact with the constricted end

of the capillary. The pyknometer is removed from the thermostat,

wiped dry with a taintless cloth. Then this filled pyknometer is

placed in an electronic balance and the reading is taken accurately.

The experiment is conducted for temperatures 25°C, 30°C, 35°C, 40°C

and 45°C in a phased manner keeping the temperature steady. Water

is then removed and the pyknometer is dried. The pyknometer IS

then filled with the experimental liquid and the whole process IS

repeated as before.

5.6.2 THEORY

Let WlI Wz and W3 be the mass of empty pyknometer,

pyknometer filled with water and that with the liquid respectively at

the same temperature. The density of the liquid p at this temperature

is given by the formula,

(5.7)

This method is very accurate SInce mass correct to

±O.1mg can be measured using the electronic balance. The accuracy

of the thermometer used in the constant temperature bath is ± O.1K.

[Robert, 2002]

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5.6.3 PRECAUTIONS

The following precautions are to be taken

experiment with pyknometer.

1. Care must be taken to see that the pyknometer is filled

slowly without air bubbles. An aspirator can be used for

the same.

2. Enough time should be given in the constant temperature

bath so that the liquid inside the pyknometer acquires the

same temperature of the bath.

3. The outer surface of the pyknometer should be dried well

before taking weights.

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