Experiment No.

1
BATCH REACTOR
AIM OF THE EXPERIMENT:
To find the activation energy for the system of sodium hydroxide and ethyl acetate in
a batch reactor
PRINCIPLE:
In batch reactors, no material is added or withdrawn during its operation. The
reactants are charged into the reactor before the commencement of the reaction and the
products are taken out after the operation. Due to good agitation, the same species
composition and temperature exists everywhere in the reacting fluid. Hence at any instant, the
same reaction rates prevail throughout the entire reactor volume and the composition
throughout the reaction remains uniform since no fluids enter or leave the reaction mixture.
For a given reaction A + B

C + D in a batch reactor,

We have,
XA/ (1-XA) = KCAot
Where
CAo = Initial concentration of A
K = rate constant
t = reaction time (min)
Arhennius law is given by K = Koe-E/(RT)
Where
K = Rate constant
Ko = frequency or pre-exponential factor
E = Activation energy (J/mol)
R = Gas constant
T = Temperature (K)
From Arhennius law:
(i)
(ii)
(iii)
(iv)

A plot of lnK v/s 1/T gives a straight line with large slope for large E and small
slope for small E.
Reactions with high Es are highly temperature sensitive reactions whereas
reactions with low Es are relatively temperature insensitive.
Any given reaction is much more temperature sensitive at low temperature than at
a high temperature.
The value of Ko does not affect the temperature sensitivity.
1

Arhennius Law is thus a good approximation to the temperature dependency of both

collision and transition state theories.
EXPERIMENTAL SETUP

Figure (i) showing a batch reactor

PROCEDURE:
1. Solutions of sodium hydroxide, hydrochloric acid and ethyl acetate are prepared at a
normality of 0.05 N.
2. After the preparation of the solution, HCl is standardised with sodium carbonate using
methyl orange as indicator and NaOH is standardised with HCl using methyl orange
as indicator.
3. 60 ml of NaOH and 60 ml of ethyl acetate are mixed well and time is noted for the
reaction between NaOH and ethyl acetate.
4. Every 5 minutes, 20 ml of reaction mixture is taken out and mixed with 10 ml of HCl
to arrest the reaction.
5. The arrested reaction mixture is titrated against 0.05 N NaOH using phenolphthalein
indicator.
6. Similarly, the experiment is repeated for 10 minutes, 15 minutes, 20 minutes, 25
minutes and the values are all calculated.

Model graphs:
(i) XA /(1 XA) v/s Time (t)

(ii) lnK v/s 1/T

RESULT:
The activation energy for the system of NaOH and ethyl acetate in batch reactor is

Experiment No.2
EXCESS PROPERTY DETERMINATION
AIM
To determine the partial molar volume and excess molar volume of a non-ideal
mixture at atmospheric pressure and ambient temperature.
MATERIALS REQUIRED
Benzene, acetic acid, water, specific gravity bottle, electronic weighing machine,
beaker and measuring cylinder
THEORY
Excess functions are thermodynamic properties of solutions which are in excess of
those of an ideal solution at the same temperature, pressure and composition. If M represents
the molar value of any extensive property (V, U, H, S, G) then an excess property represented
by ME is defined as the difference between the actual property value of a solution and the
value it would have as an ideal solution at the same temperature, pressure and composition.
ME = M Mid where Mid represents the molar value of any extensive property if the solution
was behaving ideally.
If the thermodynamic property is the volume, then the excess volume is given by
VE = V Vid Excess functions may be positive or negative. When the excess property of a
solution is greater than zero, the solution is said to exhibit positive deviations from ideality. If
it is less than zero, then solution is said to exhibit negative deviations from ideality. For a
component in an ideal solution, the partial molar property is same as the pure component
molar property. If the mixture behaves as a non-ideal solution, then the partial molar property
will be greater or less than pure component molar property.
The solution property that is formed by mixing two or more individual components
may be ideal or non-ideal and the resulting solution may be ideal or non-ideal. The excess
property determination is very much useful in determining the property of mixing and is used
in solution property determination.

PROCEDURE:
System: Benzene Acetic acid
Different volumes of benzene (2cc, 4cc 18cc) are taken and mixed with acetic acid
to form a binary mixture. The total volume of the mixture is kept constant at 25cc. The
density of the mixture is found by measuring the weight of the mixture in a specific gravity
bottle of 25cc. The density of pure components and water is also found out. From the density
of pure components, the individual weight, moles and mole fractions of the binary mixture
are found. From the density of mixture, specific volume of mixture is calculated.
A graph of molar volume Vs composition of benzene is to be drawn. From the graph
at different composition (say 0.1, 0.2 . 1) determine partial molar property by Tangent
Intercept method.
t each of the selected liquid composition, find V1E by using equation

here
V1, V2

1,

V1E

V1

V2E

V2

= partial molar volume of components at specified mole fractions.

pure component molar volumes

The excess volume of mixture can be found by using the formula
VE = x1V1E x2V2E
A graph between VE,

and x1, mole fraction of first component, is drawn to

find the partial molar excess volume at infinite dilutions.

GRAPHS

10

11

12

RESULT AND CONCLUSION

Graph shows that the given system benzene-acetic acid exhibits ________________
from ideality at ambient temperature and pressure.

13

Experiment No.3
FERMENTATION-GROWTH CURVE
AIM
To study the growth pattern of the given E-coli strain.
MATERIALS REQUIRED
E-coli strain, conical flask, inoculation loop, cotton, centrifuge tubes, peptone, beef
extract, NaCl, phosphate buffer
THEORY
Fermentation in microbiology is used to describe any process for the production of a
product by means of the mass culture of a microorganism.
The product can either be:
1. The cell itself: referred to as biomass production.
2. A microorganisms own metabolite: referred to as a product from a natural or
genetically improved strain.
3. A microorganisms foreign product: referred to as a product from recombinant DNA
technology or genetically engineered strain, i.e. recombinant strain.

Bacterial growth in batch culture can be modelled with four different phases: lag phase (A),
exponential or log phase (B), stationary phase (C) and death phase (D).
1. During lag phase, bacteria adapt themselves to growth conditions. It is the period
where the individual bacteria are maturing and not yet able to divide. During the lag
phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules
occurs.
2. Exponential phase (sometimes called the log phase or the logarithmic phase) is a
period characterized by cell doubling. The number of new bacteria appearing per unit
time is proportional to the present population. If growth is not limited, doubling will
continue at a constant rate so both the number of cells and the rate of population
increase doubles with each consecutive time period. For this type of exponential
14

growth, plotting the natural logarithm of cell number against time produces a straight
line. The slope of this line is the specific growth rate of the organism, which is a
measure of the number of divisions per cell per unit time. The actual rate of this
growth (i.e. the slope of the line in the figure) depends upon the growth conditions,
which affect the frequency of cell division events and the probability of both daughter
cells surviving. Under controlled conditions, cyanobacteria can double their
population four times a day. Exponential growth cannot continue indefinitely,
however, because the medium is soon depleted of nutrients and enriched with wastes.
3. The stationary phase is due to a growth-limiting factor; this is mostly depletion of a
nutrient, and/or the formation of inhibitory products such as organic acids. An
awkward but unfortunately widespread explanation is that the stationary phase results
from a situation in which growth rate and death rate have the same values (newly
formed cells per time = dying cells per time) but this is not logical, and it is better to
forget this. Such an explanation would not be in accordance with the observed
substrate depletion and also could never explain the rather smooth, horizontal
linear part of the curve during the stationary phase. Death of cells as a function of
time is rather unpredictable and very difficult to explain. Another not really logical
explanation of the stationary phase is that there isnt any more enough space for the
cells. However, under the microscope you will see that there is still plenty of water
between the cells.Only in an agar colony with densely packed cells space is obviously
limiting.
4. At death phase, bacteria run out of nutrients and die. This basic batch culture growth
model draws out and emphasizes aspects of bacterial growth which may differ from
the growth of macrofauna. It emphasizes clonality, asexual binary division, the short
development time relative to replication itself, the seemingly low death rate, the need
to move from a dormant state to a reproductive state or to condition the media, and
finally, the tendency of lab adapted strains to exhaust their nutrients. In reality, even
in batch culture, the four phases are not well defined. The cells do not reproduce in
synchrony without explicit and continual prompting (as in experiments with stalked
bacteria) and their exponential phase growth is often not ever a constant rate, but
instead a slowly decaying rate, a constant stochastic response to pressures both to
reproduce and to go dormant in the face of declining nutrient concentrations and
increasing waste concentrations.

15

Batch culture is the most common laboratory growth method in which bacterial growth is
studied.
PROCEDURE
A. PREPARATION OF THE MEDIUM:
250 ml of the medium is prepared by using the following components.
Peptone - 5.0g/l
Beef extract - 3.0g/l
NaCl - 0.2g/l
pH - 7.0
Distilled water 1000ml

(i)

The above components are weighed and mixed well in 250ml of distilled water.

(ii)

pH of the medium is adjusted to 7.0.

(iii)

50ml of the medium is transferred into four 250ml conical flask and plugged with
cotton.

(iv)

The medium is sterilized in an autoclave (120oC, 14 psi pressure)

B. INOCULATION
(i)

The sterilized medium is cooled and one loopful of E-coli strain from stock culture is
inoculated into one 50ml flask.

(ii)

The inoculated flask is allowed to grow for 24hr in a reciprocating shaker at 250 rpm.

(iii)

After 24 hrs, 8ml of the inoculum is transferred from the pre-grown culture into four
flask containing 50 ml sterilized medium.

(iv)

The four flasks at 30 c are incubated in a reciprocating shaker.

(v)

The sample is analysed for every 24 hrs for determining the cell mass by centrifuging
known volume of grown culture at 10,000 rpm for 10 minutes.

C. DETERMINATION OF CELL MASS

(i)

The empty centrifuge tube is weighed before centrifuging.

(ii)

After centrifuging, the supernatant liquid is decanted and the wet cell mass is
weighed.

(iii)

The cell mass is calculated using the following formula

16

Cell mass = (weigh of the centrifuge tube with cell mass after centrifuging) - (empty
weigh of the centrifuge)

RESULT
A Plot between time (hr) Vs cell mass (g/I) gives the following graph,
Where;
AB

Lag phase

BC

Exponential phase

CD

Stationary phase

DE -

Declining phase

17

Experiment No.4
GAS CHROMATOGGRAPHY
AIM
Performance analysis of gas chromatography
THEORY
Gas chromatography (GC) is a common type of chromatography used in analytical
chemistry for separating and analyzing compounds that can be vaporized without
decomposition. Typical uses of GC include testing the purity of a particular substance or
separating the different components of a mixture, the relative amounts of which can also be
determined. In some situations, GC may help in identifying a compound. In preparative
chromatography, GC can also be used to prepare pure compounds from a mixture.
In gas chromatography, the mobile phase (or moving phase) is a carrier gas, usually
an inert gas such as helium or a non-reactive gas such as nitrogen. The stationary phase is a
microscopic layer of liquid or polymer on an inert solid support inside a piece of glass or
metal tubing called a column. The instrument used to perform gas chromatography is called a
gas chromatograph (or aerograph or gas separator).The gaseous compounds being analyzed
interact with the walls of the column which is coated with the stationary phase. This causes
each compound to elute at a different time, known as the retention time of the compound. The
comparison of retention times is what gives GC its analytical usefulness.
Gas chromatography is in principle similar to column chromatography, as well as
other forms of chromatography, such as HPLC, TLC, etc. but it has several notable
differences. Firstly, the process of separating the compounds in a mixture is carried out
between a liquid stationary phase and a gas mobile phase, whereas in column
chromatography the stationary phase is a solid and the mobile phase is a liquid. Hence the
full name of the procedure is "Gasliquid chromatography", referring to the mobile and
stationary phases, respectively. Secondly, the column through which the gas phase passes is
located in an oven where the temperature of the gas can be controlled, whereas column
chromatography typically has no such temperature control. Thirdly, the concentration of a
compound in the gas phase is solely a function of the vapor pressure of the gas.
18

Gas chromatography is also similar to fractional distillation, since both processes

separate the components of a mixture primarily based on boiling point or vapor pressure
differences. However, fractional distillation is typically used to separate components of a
mixture on a large scale, whereas GC can be used on a much smaller scale (i.e. micro-scale).
Gas chromatography is also sometimes known as vapor-phase chromatography (VPC)
or gasliquid partition chromatography (GLPC). These alternative names, as well as their
respective abbreviations, are frequently used in scientific literature. Strictly speaking, GLPC
is the most correct terminology, and is thus preferred by many authors.

GC ANALYSIS
A gas chromatograph basically is a chemical analysis instrument used for separating
chemicals in a given sample. It uses a flow-through narrow tube known as the column,
through which different chemical constituents of a sample pass in a gas stream (carrier gas or
mobile phase) at different rates depending on their various chemical and physical properties
and their interaction with a specific column filling, called the stationary phase. As the
chemicals exit the end of the column, they are detected and identified electronically. The
function of the stationary phase in the column is to separate different components, causing
each one to exit the column at a different time (retention time). Other parameters that can be
used to alter the order or time of retention are the carrier gas flow rate, column length and the
temperature.
PROCEDURE:
In a GC analysis, a known volume of gaseous or liquid analyte is injected into the
entrance (head) of the column, usually using a microsyringe (or solid phase microextraction
fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules
through the column, this motion is inhibited by the adsorption of the analyte molecules either
onto the column walls or onto the packing materials in the column. The rate at which the
molecules progress along the column depends on the strength of adsorption, which in turn
depends on the type of molecule and on the stationary phase materials. Since each type of
19

molecule has a different rate of progression, the various components of the analyte mixture
are separated as they progress along the column and reach the end of the column at different
times (retention time). A detector is used to monitor the outlet stream from the column. Thus
the time at which each component reaches the outlet and the amount of that component can
be determined. Generally, substances are identified qualitatively by the order in which they
elute from the column and by the retention time of the analyte in the column.

Figure: Schematic representation of a gas chromatogram

Autosamplers
Autosamplers provide the means to introduce a sample automatically into the inlets.
Manual insertion of the sample is possible but is no longer common. Automatic insertion
provides better reproducibility and time-optimization.
Inlets
The column inlet (or injector) provides the means to introduce a sample into a
continuous flow of carrier gas. The inlet is a piece of hardware attached to the column head.
Carrier gas

20

The choice of carrier gas (mobile phase) is important as hydrogen has a larger range
of flow rates that are comparable to helium in efficiency. However, helium may be more
efficient and provide the best separation if flow rates are optimized. Helium is nonflammable, and works with a greater number of detectors. Therefore, helium is the most
common carrier gas used.
Detectors
The most commonly used detectors are the flame ionization detector (FID) and the
thermal conductivity detector (TCD). Both are sensitive to a wide range of components, and
both work over a wide range of concentrations. While TCDs are essentially universal and can
be used to detect any component other than the carrier gas (as long as their thermal
conductivities are different from that of the carrier gas at detector temperature), FIDs are
sensitive primarily to hydrocarbons, and are more sensitive to them than TCD. However, an
FID cannot detect water. Both detectors are also quite robust. Since TCD is non-destructive,
it can be operated in series before an FID (destructive), thus providing complementary
detection of the same analytes.
Stationary compound selection
The polarity of the solute is crucial for the choice of stationary compound, which in
an optimal case would have a similar polarity as the solute. Common stationary phases in
open

tubular

columns

are

cyanopropylphenyl

dimethyl

polysiloxane,

carbowaxpolyethyleneglycol, biscyanopropylcyanopropylphenylpolysiloxane and diphenyl

dimethyl polysiloxane.
Inlet types and flow rates
The choice of inlet type and injection technique depends on if the sample is in liquid,
gas, adsorbed, or solid form, and on whether a solvent matrix is present that has to be
vaporized. Dissolved samples can be introduced directly onto the column via an injector, if
the conditions are well known; gaseous samples (e.g., air cylinders) are usually injected using
a gas switching valve system; adsorbed samples (e.g., on adsorbent tubes) are introduced
using either an external (on-line or off-line) desorption apparatus such as a purge-and-trap
system, or are desorbed in the injector.

21

Sample size and injection technique

The real chromatographic analysis starts with the introduction of the sample onto the
column. The injection system in the capillary gas chromatograph should fulfill the following
two requirements:
1. The amount injected should not overload the column.
2. The width of the injected plug should be small compared to the spreading due to the
chromatographic process. Failure to comply with this requirement will reduce the
separation capability of the column.

Some general requirements which a good injection technique should fulfil are:

It should be possible to obtain the columns optimum separation efficiency.

It should allow accurate and reproducible injections of small amounts of

representative samples.

It should induce no change in sample composition. It should not exhibit

discrimination based on differences in boiling point, polarity, concentration or
thermal/catalytic stability.

It should be applicable for trace analysis as well as for undiluted samples.

Column selection
The choice of column depends on the sample and the active measured. The main
chemical attribute regarded when choosing a column is the polarity of the mixture, but
functional groups can play a large part in column selection. The polarity of the sample must
closely match the polarity of the column stationary phase to increase resolution and
separation while reducing run time. The separation and run time also depends on the film
thickness (of the stationary phase), the column diameter and the column length.

22

Figure showing a gas chromatography oven, open to show a capillary column

Column temperature and temperature program
The column(s) in a GC are contained in an oven, the temperature of which is precisely
controlled electronically. The rate at which a sample passes through the column is directly
proportional to the temperature of the column. The higher the column temperature, the faster
the sample moves through the column. However, the faster a sample moves through the
column, the less it interacts with the stationary phase, and the less the analytes are separated.
In general, the column temperature is selected to compromise between the length of
the analysis and the level of separation. A method which holds the column at the same
temperature for the entire analysis is called "isothermal." Most methods, however, increase
the column temperature during the analysis, the initial temperature, rate of temperature
increase (the temperature "ramp") and final temperature is called the "temperature
program."A temperature program allows analytes that elute early in the analysis to separate
adequately, while shortening the time it takes for late-eluting analytes to pass through the
column.
Data reduction and analysis
Qualitative analysis
Generally chromatographic data is presented as a graph of detector response (y-axis)
against retention time (x-axis), which is called a chromatogram. This provides a spectrum of
23

peaks for a sample representing the analytes present in a sample eluting from the column at
different times. Retention time can be used to identify analytes if the method conditions are
constant. Also, the pattern of peaks will be constant for a sample under constant conditions
and can identify complex mixtures of analytes. In most modern applications however the GC
is connected to a mass spectrometer or similar detector that is capable of identifying the
analytes represented by the peaks.
Quantitative analysis
The area under a peak is proportional to the amount of analyte present in the
chromatogram. By calculating the area of the peak using the mathematical function of
integration, the concentration of an analyte in the original sample can be determined.
Concentration can be calculated using a calibration curve created by finding the response for
a series of concentrations of analyte, or by determining the relative response factor of an
analyte. The relative response factor is the expected ratio of an analyte to an internal standard
(or external standard) and is calculated by finding the response of a known amount of analyte
and a constant amount of internal standard (a chemical added to the sample at a constant
concentration, with a distinct retention time to the analyte).In most modern GC-MS systems,
computer software is used to draw and integrate peaks, and match MS spectra to library
spectra.
Application
In general, substances that vaporize below 300 C (and therefore are stable up to that
temperature) can be measured quantitatively. The samples are also required to be salt-free;
they should not contain ions. Very minute amounts of a substance can be measured.

24

Experiment No.5
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
AIM
Performance analysis of HPLC
MATERIALS REQUIRED
Resorcinol, HPLC setup, ethanol for HPLC, distilled water for HPLC
THEORY
High-performance liquid chromatography (sometimes referred to as high-pressure
liquid chromatography) HPLC, is a chromatographic technique used to separate a mixture of
compounds in analytical chemistry and biochemistry with the purpose of identifying,
quantifying and purifying the individual components of the mixture. HPLC is also considered
an instrumentation technique of analytical chemistry, instead of a gravimetric technique.
HPLC has many uses including medical (e.g. detecting vitamin D concentrations in blood
serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g.
purifying substances from a complex biological sample, or separating similar synthetic
chemicals from each other), and manufacturing (e.g. pharmaceutical quality control). HPLC
can alternatively be described as a mass transfer involving adsorption.
HPLC relies on the pressure of mechanical pumps on a liquid solvent to load a sample
mixture onto a separation column, in which the separation occurs. A HPLC separation
column is filled with solid particles (e.g. silica, polymers, or sorbents), and the sample
mixture is separated into compounds as it interacts with the column particles. HPLC
separation is influenced by the liquid solvents condition (e.g. pressure, temperature),
chemical interactions between the sample mixture and the liquid solvent (e.g. hydrophobicity,
protonation, etc), and chemical interactions between the sample mixture and the solid
particles packed inside of the separation column (e.g. Ligand affinity, ion exchange, etc...).
HPLC is distinguished from ordinary liquid chromatography because the pressure of
HPLC is relatively high, while ordinary liquid chromatography typically relies on the force of

25

gravity to provide pressure. Due to the higher pressure separation conditions of HPLC, HPLC
columns have relatively small internal diameter (e.g. 4.6 mm), are short (e.g. 25 mm), and
packed more densely with smaller particles, which helps achieve finer separations of a
sample mixture than ordinary liquid chromatography can. This gives HPLC superior
resolving power when separating mixtures, which is why it is a popular chromatographic
technique.
The schematic of an HPLC instrument typically includes a sampler by which the
sample mixture is injected into the HPLC, one or more mechanical pumps for pushing liquid
through a tubing system, a separation column, a digital analyst detector (e.g. a UV/Vis, or a
photodiode array (PDA)) for qualitative or quantitative analysis of the separation, and a
digital microprocessor for controlling the HPLC components (and user software). Many
different types of columns are available, varying in size, and in the type (i.e. chemistry) of
solid packed particle types available. Some models of mechanical pumps in a HPLC
instrument can also mix multiple liquids together, and the recipe or gradient of those liquids
can modify the chemical interactions that occur in HPLCs column, and thereby modify the
chemical separation of the mixture.
HPLC separations have theoretical parameters and equations to describe the
separation of components into signal peaks when detected by instrumentation such as by a
UV detector or mass spectrometer. The parameters are practical in the sense that they are not
predicted, but are measurements of HPLC chromatograms. They are analogous to the
calculation of retention factor for a paper chromatography separation, but describe how well
HPLC separates a mixture into two or more components that are detected as peaks (bands) on
a chromatograms. The HPLC parameters are the: efficiency factor (N), the retention factor
(kappa prime), and the separation factor (alpha). Together the factors are variables in a
resolution equation, which describes how well two components' peaks separated or
overlapped each other. These parameters are mostly only used for describing HPLC reversed
phase and HPLC normal phase separations, since those separations tend to be more subtle
than other HPLC modes (e.g. ion exchange and size exclusion).
Void volume is the amount of space in a column that is occupied by solvent. It is the
space within the column that is outside of the column's internal packing material. Void
volume is measured on a chromatogram as the first component peak detected, which is
usually the solvent that was present in the sample mixture; ideally the sample solvent flows
26

through the column without interacting with the column, but is still detectable as distinct
from the HPLC solvent. The void volume is used as a correction factor among other factors.
Efficiency factor (N) practically measures how sharp component peaks on the
chromatogram are, as ratio of the component peak's area ("retention time") relative to the
width of the peaks at their widest point (at the baseline). Peaks that are tall, sharp, and
relatively narrow indicate that separation method efficiently removed a component from a
mixture; high efficiency. Efficiency is very dependent upon the HPLC column and the HPLC
method used. Efficiency factor is synonymous with plate number, and the 'number of
theoretical plates'.
Retention factor (kappa prime) measures how long a component of the mixture stuck
to the column, measured by the area under the curve of its peak in a chromatogram (since
HPLC chromatograms are a function of time). Each chromatogram peak will have its own
retention factor (e.g. kappa1 for the retention factor of the first peak). This factor may be
corrected for by the void volume of the column.
Separation factor (alpha) is a relative comparison of how well two neighbouring
components of the mixture were separated (i.e. two neighbouring bands on a chromatogram).
This factor is defined in terms of a ratio of the retention factors of a pair of neighbouring
chromatogram peaks, and may also be corrected for by the void volume of the column. The
greater the separation factor value is over 1.0, the better the separation, until about 2.0
beyond which an HPLC method is probably not needed for separation.
Resolution equations relate the three factors such that high efficiency and separation
factors improve the resolution of component peaks in a HPLC separation.
The internal diameter (ID) of an HPLC column is an important parameter that
influences the detection sensitivity and separation selectivity in gradient elution. It also
determines the quantity of analyte that can be loaded onto the column. Larger columns are
usually seen in industrial applications, such as the purification of a drug product for later use.
Low-ID columns have improved sensitivity and lower solvent consumption at the expense of
loading capacity.

27

EXPERIMENTAL SETUP

PROCEDURE:
The sample to be separated and analyzed is introduced in a discrete small volume into
the stream of mobile phase percolating through the column. The components of the sample
move through the column at different velocities which are functions of specific physical or
chemical interactions with the stationary phase. The velocity of each component depends on
its chemical nature, on the nature of the stationary phase and on the composition of the
mobile phase. The time at which a specific analyte elutes (emerges from the column) is called
the retention time. The retention time measured under particular conditions is considered the
identifying characteristic of a given analyte. The use of smaller particle size packing
materials requires the use of higher operational pressure (backpressure) and typically
improves chromatographic resolution (i.e. the degree of separation between consecutive
analytes emerging from the column). Common mobile phases used include any miscible
combination of water with various organic solvents (the most common are acetonitrile and
methanol). Some HPLC techniques use water free mobile phases. The aqueous component of
the mobile phase may contain buffers, acids (such as formic, phosphoric or trifluoroacetic
acid) or salts to assist in the separation of the sample components. The composition of the

28

mobile phase may be kept constant (isocratic elution mode) or varied (gradient elution mode)
during the chromatographic analysis.
In gradient elution the composition of the mobile phase is varied typically from low to
high eluting strength. The eluting strength of the mobile phase is reflected by analyte
retention times with high eluting strength producing fast elution (=short retention times).
The composition of the mobile phase depends on the intensity of interactions between
analytes and stationary phase (e.g. hydrophobic interactions in reversed-phase HPLC).
Depending on their affinity for the stationary and mobile phases analytes partition between
the two during the separation process taking place in the column. This partitioning process is
similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise.
In this example, using a water/acetonitrile gradient, more hydrophobic components will elute
(come off the column) late, once the mobile phase gets more concentrated in acetonitrile (i.e.
in a mobile phase of higher eluting strength).
The choice of mobile phase components, additives (such as salts or acids) and
gradient conditions depend on the nature of the column and sample components. Often a
series of trial runs are performed with the sample in order to find the HPLC method which
gives the best separation.

29

OBSERVATION AND CALCULATION

Result
We get the peak at 6.755 mintues, which is the retention time.

30

Experiment No.6
TENSOMETER
AIM
To determine the tensile strength of given sample using a tensile testing instrument
THEORY
Tensile testing also known as tension testing is a fundamental materials science test in
which a sample is subjected to uniaxial tension until failure. The results from the test are
commonly used to select a material for an application, for quality control, and to predict how
a material will react under other types of forces. Properties that are directly measured via a
tensile test are ultimate tensile strength, maximum elongation and reduction in area. From
these measurements the following properties can also be determined: Young's modulus,
Poisson's ratio, yield strength, and strain-hardening characteristics.
A tensometer is a device used to evaluate the Young's modulus (how much it stretches
under stress) of a material and other tensile properties of materials, such as tensile strength. It
is usually a universal testing machine loaded with a sample between 2 grips that are either
adjusted manually or automatically to apply force to the specimen. The machine works either
by driving a screw or by hydraulic ram. The latter have the great advantage of being able to
create much more complex loading patterns, such as the cyclical loads needed for
measurement of fatigue strength. Machines can also be equipped with environmental
chambers for testing at different temperatures or variable humidity.

A tensometer is a device used to determine a material's response to varying strains,

called loads. The amount of stretch that a material has when it is under strain provides
important

information

about

the

materials

tensile

strength

and

fatigue

strength. Tensometer devices are routinely used in the manufacturing industry to ensure that
parts meet necessary strength and endurance requirements.
Tensometer devices consist of two grips that hold a section of test material in place. These
grips are then used to apply a tensile or compression force, called a load, to the test piece.
31

Tensometer instruments can create the force through the use of either a screw or a hydraulic
ram, which are powered by mechanical or electrical means.

The test process involves placing the test specimen in the testing machine and
applying tension to it until it fractures. During the application of tension, the elongation of the
gauge section is recorded against the applied force. The data is manipulated so that it is not
specific to the geometry of the test sample.
The elongation measurement is used to calculate the engineering strain, , using the
following equation:

Where L is the change in gauge length, L0 is the initial gauge length, and L is the
final length. The force measurement is used to calculate the engineering stress, , using the
following equation:

Where F is the force and A is the cross-section of the gauge section. The machine
does these calculations as the force increases, so that the data points can be graphed into a
stress-strain curve.
Material to be tested must be cut to a specific shape so as to fit the grips, most usually
in the form of a dog-bone shape when flat sheet is being tested. The sheet is cut or machined
to shape, and great care is needed to create a smooth edge. If defects are left, the result may
be premature failure from the defect, thus underestimating tensile strength. Different shaped
samples need different grip designs to achieve their objective. Fibres for example, require rod
grips around which fibre can be wound prior to straining. Product tests may even need special
grips to be designed owing to the shape variability of complex products.
The measurements taken are load and extension and a tensometer gives directly the
load extension curve, and not the stress-strain curve. However, the stress-strain curve can be
calculated from the cross-sectional area and the original length of the specimen. Such a curve

32

can be labelled with specific identifiable point such as elastic limit, yield point and fracture.
A tensometer can be either electronic or manual, the latter being with a handle to apply force.

Figure: Stress Strain Curve

33

Experimental Setup

Figure: Experimental setup of a tensometer

34

Experiment No.7
UV SPECTROSCOPY
AIM
Performance analysis of UV spectroscopy
THEORY
Ultravioletvisible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or
UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultravioletvisible spectral region. This means it uses light in the visible and adjacent (near-UV and nearinfrared (NIR)) ranges. The absorption or reflectance in the visible range directly affects the
perceived color of the chemicals involved. In this region of the electromagnetic spectrum,
molecules undergo electronic transitions.
Molecules containing -electrons or non-bonding electrons (n-electrons) can absorb
the energy in the form of ultraviolet or visible light to excite these electrons to higher antibonding molecular orbitals. The more easily excited the electrons the longer the wavelength
of light it can absorb.
The Beer-Lambert law states that the absorbance of a solution is directly proportional
to the concentration of the absorbing species in the solution and the path length Thus, for a
fixed path length, UV/Vis spectroscopy can be used to determine the concentration of the
absorber in a solution. It is necessary to know how quickly the absorbance changes with
concentration. This can be taken from references (tables of molar extinction coefficients), or
more accurately, determined from a calibration curve.

whereA is the measured absorbance,

wavelength,

is the intensity of the incident light at a given

is the transmitted intensity, L the path length through the sample, and c the

concentration of the absorbing species. For each species and wavelength, is a constant
known as the molar absorptivity or extinction coefficient.

35

A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an

analyte gives a response assumed to be proportional to the concentration. For accurate results,
the instrument's response to the analyte in the unknown should be compared with the
response to a standard; this is very similar to the use of calibration curves. The response (e.g.,
peak height) for a particular concentration is known as the response factor.
The Beer-Lambert law has implicit assumptions that must be met experimentally for it
to apply otherwise there is a possibility of deviations from the law to be observed. For
instance, the chemical makeup and physical environment of the sample can alter its extinction
coefficient. The chemical and physical conditions of a test sample therefore must match
reference measurements for conclusions to be valid
The instrument used in ultraviolet-visible spectroscopy is called a UV/Vis
spectrophotometer. It measures the intensity of light passing through a sample ( ), and
compares it to the intensity of light before it passes through the sample (

). The ratio

is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A
is

based on the transmittance:

The UV-visible spectrophotometer can also be configured to measure reflectance. In

this case, the spectrophotometer measures the intensity of light reflected from a sample ( ),
and compares it to the intensity of light reflected from a reference material (
white tile). The ratio

) (such as a

is called the reflectance, and is usually expressed as a percentage

(%R).
The basic parts of a spectrophotometer are a light source, a holder for the sample, a
diffraction grating in a monochromator or a prism to separate the different wavelengths of
light, and a detector. The radiation source is often a Tungsten filament (300-2500 nm), a
deuterium arc lamp, which is continuous over the ultraviolet region (190-400 nm), Xenon arc
lamp, which is continuous from 160-2,000 nm; or more recently, light emitting diodes (LED)
for the visible wavelengths. The detector is typically a photomultiplier tube, a photodiode, a
photodiode array or a charge-coupled device (CCD). Single photodiode detectors and
photomultiplier tubes are used with scanning monochromators, which filter the light so that
36

only light of a single wavelength reaches the detector at one time. The scanning
monochromator moves the diffraction grating to "step-through" each wavelength so that its
intensity may be measured as a function of wavelength. Fixed monochromators are used with
CCDs and photodiode arrays. As both of these devices consist of many detectors grouped
into one or two dimensional arrays, they are able to collect light of different wavelengths on
different pixels or groups of pixels simultaneously.

Figure: Single-beam UV/Vis spectrophotometer

A spectrophotometer can be either single beam or double beam. In a single beam
instrument (such as the Spectronic20), all of the light passes through the sample cell.

must

be measured by removing the sample. In a double-beam instrument, the light is split into two
beams before it reaches the sample. One beam is used as the reference; the other beam passes
through the sample. The reference beam intensity is taken as 100% Transmission (or 0
Absorbance), and the measurement displayed is the ratio of the two beam intensities. Some
double-beam instruments have two detectors (photodiodes), and the sample and reference
beam are measured at the same time. In other instruments, the two beams pass through a
beam chopper, which blocks one beam at a time. The detector alternates between measuring
the sample beam and the reference beam in synchronism with the chopper. There may also be
one or more dark intervals in the chopper cycle. In this case, the measured beam intensities
may be corrected by subtracting the intensity measured in the dark interval before the ratio is
taken.
Samples for UV/Vis spectrophotometry are most often liquids, although the
absorbance of gases and even of solids can also be measured. Samples are typically placed in
37

a transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape, commonly
with an internal width of 1 cm. (This width becomes the path length,

, in the Beer-Lambert

law.) Test tubes can also be used as cuvettes in some instruments. The type of sample
container used must allow radiation to pass over the spectral region of interest. The most
widely applicable cuvettes are made of high quality fused silica or quartz glass because these
are transparent throughout the UV, visible and near infrared regions. Glass and plastic
cuvettes are also common, although glass and most plastics absorb in the UV, which limits
their usefulness to visible wavelengths. Specialized instruments have also been made. These
include attaching spectrophotometers to telescopes to measure the spectra of astronomical
features. UV-visible microspectrophotometers consist of a UV-visible microscope integrated
with a UV-visible spectrophotometer.
Experimental setup

Figure: Experimental setup of a UV spectrophotometer

38

EXPERIMENT NO.8

SCANNING ELECTRON MICROSCOPE

AIM
To find out surface characteristics of
(i)

Copper coated activated alumina

(ii)

Sisal fibre

using SEM (Scanning Electron Microscope)

MATERIALS REQUIRED
Copper coated activated alumina, sisal fibre
THEORY
A scanning electron microscope (SEM) is a type of electronmicroscope that produces
images of a sample by scanning it with a focused beam of electrons. The electrons interact
with the electrons in the sample, producing various signals that can be detected and that
contain information about the sample's surface topography and composition. The electron
beam is generally scanned in a rasterscan pattern, and the beam's position is combined with
the detected signal to produce an image. SEM can achieve resolution better than 1 nanometre.
The types of signals produced by a SEM include secondary electrons, back-scattered
electrons (BSE), characteristic X-rays, light (cathodoluminescence), specimen current and
transmitted electrons. Secondary electron detectors are common in all SEMs, but it is rare
that a single machine would have detectors for all possible signals. The signals result from
interactions of the electron beam with atoms at or near the surface of the sample. In the most
common or standard detection mode, secondary electron imaging or SEI, the SEM can
produce very high-resolution images of a sample surface, revealing details less than 1 nm in
size. Due to the very narrow electron beam, SEM micrographs have a large depth of field
yielding a characteristic three-dimensional appearance useful for understanding the surface
structure of a sample. A wide range of magnifications is possible, from about 10 times (about

39

equivalent to that of a powerful hand-lens) to more than 500,000 times, about 250 times the
magnification limit of the best light microscopes.
In a typical SEM, an electron beam is thermionically emitted from an electron gun
fitted with a tungsten filament cathode. Tungsten is normally used in thermionic electron
guns because it has the highest melting point and lowest vapour pressure of all metals,
thereby allowing it to be heated for electron emission, and because of its low cost.
The electron beam which typically has an energy ranging from 0.2 keV to 40 keV is
focused by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam
passes through pairs of scanning coils or pairs of deflector plates in the electron column,
typically in the final lens, which deflect the beam in the x and y axes so that it scans in a
raster fashion over a rectangular area of the sample surface.

Figure: Schematic diagram of SEM

40

When the primary electron beam interacts with the sample, the electrons lose energy
by repeated random scattering and absorption within a teardrop-shaped volume of the
specimen known as the interaction volume, which extends from less than 100 nm to around
5 m into the surface. The size of the interaction volume depends on the electron's landing
energy, the atomic number of the specimen and the specimen's density. The energy exchange
between the electron beam and the sample results in the reflection of high-energy electrons
by elastic scattering, emission of secondary electrons by inelastic scattering and the emission
of electromagnetic radiation, each of which can be detected by specialized detectors. The
beam current absorbed by the specimen can also be detected and used to create images of the
distribution of specimen current. Electronic amplifiers of various types are used to amplify
the signals, which are displayed as variations in brightness on a computer monitor (or, for
vintage models, on a cathode ray tube). Each pixel of computer videomemory is synchronised
with the position of the beam on the specimen in the microscope, and the resulting image is
therefore a distribution map of the intensity of the signal being emitted from the scanned area
of the specimen. In older microscopes image may be captured by photography from a highresolution cathode ray tube, but in modern machines image is saved to a computer data
storage.
Magnification in a SEM can be controlled over a range of up to 6 orders of magnitude
from about 10 to 100,000 times. Unlike optical and transmission electron microscopes, image
magnification in the SEM is not a function of the power of the objective lens. SEMs may
have condenser and objective lenses, but their function is to focus the beam to a spot, and not
to image the specimen. Provided the electron gun can generate a beam with sufficiently small
diameter, a SEM could in principle work entirely without condenser or objective lenses,
although it might not be very versatile or achieve very high resolution. Magnification is
therefore controlled by the current supplied to the x, y scanning coils, or the voltage supplied
to the x, y deflector plates, and not by objective lens power.
EXPERIMENTAL SETUP

41

PROCEDURE
1. The sample under observation is coated with gold if it is non-conductive. Copper
coated alumina being conductive is directly placed on the sample holder of the SEM.
2. The sample holder is fitted in the SEM.
42

3. Vacuum is created at 10-6torr with the help of rotary and diffusion pumps.
4. Electron beam is generated by the electron gun by accelerating it with a voltage from
1 KV to 30 KV.
5. Water in the chiller at 20oC is used to cool the heated part of SEM.
6. Images of required resolution, magnification and clarity are scanned and obtained.
7. Steps 1 to 6 are repeated for sisal fibre sample.
RESULT

Figure: Copper coated activated alumina

43

Figure showing the diamter of the activated alumina

44

Figure showing the surface of the activated alumi

Figure showing the diamter of the activated alumina

45

46