chapter experimental methods - inflibnet...
TRANSCRIPT
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
98
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
99
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]
100
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.
101