Name: Tahir

Roll no: 22-80088

Section: A
Submitted to: Dr. Khurram Rehman
Question no: 1
Separation procedure of belladonna
alkaloids through adsorption
chromatography

Adsorption chromatography
It is an analytical separation of chemical
mixtures based on the interaction of
adsorbate with adsorbent that is
chemicals are retained based on their
adsorption and desorption at the surface
of the support, which act as stationary
phase. The mixture of gas or liquid are to
be separated when it passes over the
adsorbent bed that absorbs different
components at specific rates.
Components for separation
Belladonna alkaloids that are atropine,
hyoscyamine and scopolamine. The
column utilizes a stationary phase and a
moving phase (1:1 cyclohexane carbon
tetrachloride).
Diagram
Separation procedure of belladonna
alkaloids through adsorption
chromatography
 Take a clean and dry
chromatographic jar.
 Add the mobile phase that is 1:1
cyclohexane carbon tetrachloride.
 Leave the jar for some time so that it
can be saturated with the solvent.
 Mark the baseline on the adsorbent.
 Spot a drop of belladonna alkaloids
with the help of capillary tube on the
strip of chromatographic plate. Allow
it to dry.
 Wait until the solvent moves from
baseline.
 Take out the TLC plate and dry it.
 Detect the center of the spot and
then the distance of spot from the
origin
 Separation can be done by comparing
the calculated Rf values with the
known Rf values of alkaloids.
 Rf = Distance moved by substance
from the origin / Distance moved by
solvent from origin.

Question no: 2
Quantification procedure for any
organic containment in water through
GCMS
GCMS
It is a technique comprising gas
chromatograph coupled to mass
spectrometer by which quantification,
identification and separation of
complex mixture of chemical
compounds occur.
Principle
Mixture separate into individual
substances when heated. The heated
gasses are streamed through the
column with an inert gas. As they
separated substance emerge from the
column opening, they flow into mass
spectrometer. Mass spectrometer
identifies compounds by the mass of
the analyte molecule.

Procedure
 Put the liquid sample (water
containing the organic compounds:
in this case vinyl chloride, carbon
tetrachloride and methylene
chloride) in gas chromatograph that
the sample is entered in the pot.
Heat it to up to 300 Celsius due to
which the material is volatilized.
 An inert gas carries the sample gas
into the column.
 Gaseous components are separated
as they flow through the column.
 The column is bound within a
special oven controlling the
temperature from -20 – 320 Celsius.
The column surface is coated with a
material and separate various
chemical compounds on the basis of
size and polarity. The components
that are more volatile and smaller
will travel through the column more
quickly than others.
 Then the components that are
separated are entered into mass
spectrometer.
 Components are blasted with
electrons causing them to break up
and turn into positively charged
ions.
 The ions are passed through an
electromagnetic field and filter
through based on mass and charge.
 Counting the number of filtered
ions, the information is sent to a
computer and a mass spectrum, a
distribution of ions of different sizes
are generated.
 At the end, they get entered into
the identifier of mass spectrometer
and their plentitudes and
quantifications are measured.

Question no:3
 Determine Lead and arsenic through
the principle of atomic absorbance
spectrometer

Atomic absorption spectroscopy

It is a technique for the quantitative
determination of compounds using the
absorption of optical radiation by free
atoms in the gaseous state.
Principle
Works on the principle that free
atoms (gasses) absorb radiation at
specific frequencies.

Procedure
 Place the sample in the atomic
absorption spectrometer.
 Place the nebulizer in the
volumetric flask containing the
sample, which will spray the
compound and burning flames
will produce due to which the
metal atoms (that in this case
are lead and arsenic) are
volatilized in flame and radiation
when passed through the flame.
 These volatized atoms which are
lead and arsenic will absorb
radiation with an energy
difference present between their
ground state and excited state.
 The number of atoms in the
ground state becomes excited
and emit energy.
 Hollow cathode lamp will be
used as light source; light will get
absorbed by the sample.
 As we know anode is always
constant which is tungsten.
 In case of lead cathode is coated
with lead metal so it is lead
coated cathode and excitation of
lead atoms produce a particular
radiation which is absorbed by
atoms in the flame.
 Monochromator analyze that
how much light was being
absorbed.
 Then detector will inform about
the results of the sample.
 The quantity of the lead or
arsenic is shown by area under
the graph.
 The more the concentration in
the graph the more light will be
absorbed.