|
Sr.no |
practicals |
|
1 |
Introduction of Colorimeter |
|
2 |
To
find out |
|
3 |
To
find out |
|
4 |
To
find out unknown concentration of sample using colorimeter. |
|
5 |
Introduction
of UV visible Spectrophotometer |
|
6 |
To
Find out |
|
7 |
Determination
of unknown concentration of pharmaceutical substance by using UV-Visible
spectroscopy |
|
8 |
Determination of ℅ content of sample using UV-Visible
spectrophotometer. |
|
|
INFRARED SPECTROSCOPY |
|
9 |
Identification of unknown compound
using IR Spectroscopy |
|
|
NUCLEAR MAGNETIC RESONANCE
SPECTROSCOPY |
|
10 |
Identification of unknown compound
using NMR Spectroscopy. Spectrum study and different functional groups
interpretation. |
|
|
MASS
SPECTROMETRY |
|
11 |
Identification of unknown compound
using mass Spectrometry also study of fragmentation and relative abundance. |
|
12 |
pH MEASUREMENT |
Introduction
COLORIMETRY:
It is a method of
analysis in which colored solutions are analyzed by measuring intensity of
transmitted light.
Colorimetry is the use of the
human eye to determine the concentration of colored species.
Spectrophotometry is the use of
instruments to make the same measurements.
It extends the range of possible measurements beyond those that can be
determined by the eye alone.
TRANSMITTANCE:
When light is passed
through a solution, a portion of light is absorbed while rest is transmitted.
This property is called transmittance.
T= I/I0 ℅T= I/I0
I=
Intensity of transmitted light
I0=
Intensity of incident light
ABSORBANCE:
Absorbance
is log of inverse of transmittance
A=log 1/T
The
lamp
emits all colors of light (i.e., white light).
The
monochromator
selects one wavelength and that wavelength is sent through the
sample.
The
detector
detects the wavelength of light that has passed through the sample.
The
amplifier
increases the signal so that it is easier to read against the background noise.
Visual
Observations – Because
colorimetry is based on inspection of materials with the human eye, it is
necessary to review aspects of visible light.
Visible
light
is the narrow range of electromagnetic waves with the wavelength of 400-700
nm
|
Observed Color of Compound
|
Color of Light Absorbed
|
Approximate Wavelength of
Light Absorbed |
|
Green |
Red |
700 nm |
|
Blue-green |
Orange-red |
600 nm |
|
Violet |
Yellow |
550 nm |
|
Red-violet |
Yellow-green |
530 nm |
|
Red |
Green |
500 nm |
|
Orange |
Blue |
450 nm |
|
Yellow |
Violet |
400 nm |
For
more than one color: the ratio of an
unknown mixture can also be determined by matching the shade of the color to
those produced from known ratios. The ratio of a mixture of red and blue
can be determined visibly by comparing the mixture to purples produced
from known ratios of red and blue.
To
find out
EQUIPMENT:
Colorimeter
SAMPLE:
Potassium per magnate
PROCEDURE:
·
Turn on equipment few min prior to use
·
Place blank or reference
solution in sample holder
·
Adjust absorbance to zero,
using monochromator or filter of lowest wavelength.
·
Now replace blank solution with
colored solution and note absorbance.
·
Repeat the same procedure for
all the available filters (405,492,546,578,660nm).
·
Observe wavelength at which
maximum absorbance obtained, which is considered to be
To
find out
EQUIPMENT:
Colorimeter
SAMPLE:
Potassium di chromate
PROCEDURE:
·
Turn on equipment few min prior to use
·
Place blank or reference
solution in sample holder
·
Adjust absorbance to zero,
using monochromator or filter of lowest wavelength.
·
Now replace blank solution with
colored solution and note absorbance.
·
Repeat the same procedure for
all the available filters (405,492,546,578,660nm).
·
Observe wavelength at which
maximum absorbance obtained, which is considered to be
To
find out unknown concentration of sample using colorimeter.
EQUIPMENT:
Colorimeter
SAMPLE:
Different
concentrations of sample (e.g 1℅, 2℅, 3℅, 4℅ and 5℅ Potassium per magnate)
Unknown concentration of sample
PROCEDURE:
·
Turn on equipment few min prior to use
·
Place blank or reference
solution in sample holder
·
Adjust absorbance to zero,
using monochromator or filter of lowest wavelength.
·
Now replace blank solution with
colored solution and note absorbance.
·
Since we are using KMnO4, we
make different concentration and repeat above mentioned procedure on all of
them and measure absorbance.
·
Now take solution of unknown
concentration and check its absorbance.
·
Now by plotting values in graph
we can determine concentration of unknown solution.
UV-VISIBLE
SPECTROSCOPY
Ultraviolet–visible
spectroscopy or ultraviolet-visible
spectrophotometry (UV-Vis or UV/Vis) refers to absorption
spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral
region. This means it uses light in the visible and adjacent (near-UV and near-infrared
(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. This technique is
complementary to fluorescence spectroscopy, in that fluorescence deals with
transitions from the excited state to the ground state, while absorption
measures transitions from the ground state to the excited state.
Principle
of ultravoilet-visible absorption:
Molecules
containing π-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 anti-bonding molecular orbitals. The more easily excited the electrons
(i.e. lower energy gap between the HOMO and the LUMO), the longer the
wavelength of light it can absorb.
HOMO: High occupied molecular orbital
LUMO: Lowest
unoccupied molecular orbital
Beer-Lambert
Law:
The method is most often used in a quantitative way to
determine concentrations of an absorbing species in solution, using the
Beer-Lambert law:
where
A is the measured absorbance, in Absorbance Units (AU), I0 is the intensity of the
incident light at a given wavelength, I is the transmitted intensity, L
the pathlength through the sample, and c the concentration of the
absorbing species. For each species and wavelength, ε is a constant known as
the molar absorptivity or extinction coefficient.
SCHEMATIC
DIAGRAM DOUBLE BEAM UV-VIS SPECTROPHOTOMETER:
Instruments for measuring the absorption of
U.V. or visible radiation are made up of the following components;
·
Sources (UV and visible)
·
Wavelength selector (monochromator)
·
Sample containers
·
Detector
·
Signal processor and readout
To Find out
EQUIPMENT:
UV-VIS Spectrophotometer.
SAMPLE:
Potassium dichromate
Procedure:
·
Turn on equipment few min prior to use
·
Make 0.01℅ w/v solution of K2Cr2O7.
·
Fill a cuvette with water and
other with sample solution
·
Set incident wavelength at
330nm
·
Bring water in liquid light
path and blank it.
·
Get colored compound solution
in light path and note the absorbance.
·
Repeat same procedure by
increasing wavelength to 10 until wavelength reaches the value of 440nm.
·
Note the values of absorbance
at various wavelengths and plot a graph of wavelength versus absorbance.
·
Determine
Determination
of unknown concentration of pharmaceutical substatance by using UV-Visible
spectroscopy.
EQUIPMENT:
UV-Visible
spectrophotometer.
PROCEDURE:
PREPARATION OF STANDARD SOLUTIONS:
Prepare various standard solutions of
colored compounds dissolving in 100ml of water. Label the beaker containing the
solution.
OPERATION
OF UV-VISIBLE SPECTROPHOTOMETER:
·
Turn the spectrophotometer ON.
·
Place in cuvettes containing
blank solution and colored compounds of
various concentrations.
·
Determine
·
Now take readings of absorbance
for all standard solutions.
·
Replace standard solution with
sample solution of unknown concentration and note the absorbance.
PLOTTING A GRAPH AND ADMINISTRATION OF UNKNOWN
CONCENTRATION:
·
Make a graph of concentration
(x-axis) versus absorbance (y-axis).
·
Plot the values of standard
solutions and test solution.
·
Find the concentration value
corresponding to the absorbance shown by the unknown solution via calibration
curve method.
Determination of ℅
content of sample using UV-Visible spectrophotometer.
EQUIPMENT:
UV-Visible
spectrophotometer
SAMPLE:
Tetracycline capsule
containing 250mg tetracycline hydrochloride
ASSAY:
Mix the contents of twenty capsules, taking care not to lose any
material when opening the capsule shells. Weigh the total contents of twenty
capsules and calculate the average amount of powder containing 250mg of
tetracycline hydrochloride. Take this accurately weighed quantity of powder
equivalent to the average weight supposed to contain 0.250g antibiotic.Transfer
this amount into 250ml volumetric flask, add about 100ml of 0.1N HCl, shake
thoroughly and make up the volume with 0.1N HCl. Tranfer 10ml of this solution
into a 100ml flask and make up the volume with water. Then transfer 10ml of the
preceding solution into a 100ml flask, add 10ml of 0.1N NaOH and make up the
volume with water. Measure the absorbance of a 1cm layer of the sample at
Calculation through dilution factor:
Stated potency of capsule= 250mg
Dilution factor= 250/250 × 10/100 ×
10/100 = 1/100
℅ content of tetracycline hydrochloride in
capsule
Absorbance of sample/ E (1℅, 1cm) × 1000 × 100
Absorbance of sample/380 ×1000 ×100 = X
Therefore, ℅ content
of tetracycline hydrochloride.
INFRARED
SPECTROSCOPY
Infrared spectroscopy (IR spectroscopy) is the spectroscopy that deals with the
infrared region of the electromagnetic spectrum that is light with a longer
wavelength and lower frequency than visible light. It covers a range of
techniques, mostly based on absorption spectroscopy. As with all spectroscopic
techniques, it can be used to identify and study chemicals. A common laboratory
instrument that uses this technique is a Fourier transform infrared (FTIR)
spectrometer.
The
infrared portion of the electromagnetic spectrum is usually divided into three
regions; the near-, mid- and far- infrared, named for their relation to the
visible spectrum. The higher-energy near-IR, approximately 14000–4000 cm−1
(0.8–2.5 μm wavelength) can excite overtone or harmonic vibrations. The
mid-infrared, approximately 4000–400 cm−1 (2.5–25 μm) may
be used to study the fundamental vibrations and associated
rotational-vibrational structure. The far-infrared, approximately
400–10 cm−1 (25–1000 μm), lying adjacent to the microwave
region, has low energy and may be used for rotational spectroscopy. The names
and classifications of these subregions are conventions, and are only loosely
based on the relative molecular or electromagnetic properties.
PRINCIPLE OF IR ABSORPTION:
IR radiation does not have enough energy to induce
electronic transitions as seen with UV. Absorption of IR is restricted to
compounds with small energy differences in the possible vibrational and
rotational states.
For a molecule to absorb IR, the
vibrations or rotations within a molecule must cause a net change in the dipole
moment of the molecule. The alternating electrical field of the radiation
(remember that electromagnetic radiation consists of an oscillating electrical
field and an oscillating magnetic field, perpendicular to each other) interacts
with fluctuations in the dipole moment of the molecule. If the frequency of the
radiation matches the vibrational frequency of the molecule then radiation will
be absorbed, causing a change in the amplitude of molecular vibration.
Molecular
vibrations
The
positions of atoms in a molecule are not fixed; they are subject to a number of
different vibrations. Vibrations fall into the two main categories of stretching
and bending.
Stretching:
Change in inter-atomic distance along bond axis
Bending: Change in angle between two bonds.
There are four types of bend:
- Rocking
- Scissoring
- Wagging
- Twisting
INSTRUMENTATION:
Identification
of unknown compund using IR Spectroscopy
Typical Infrared Absorption Frequencies
|
||||||
|
Stretching Vibrations |
Bending Vibrations |
|||||
|
Functional
Class |
Range (cm-1) |
Intensity |
Assignment |
Range (cm-1) |
Intensity |
Assignment |
|
Alkanes |
2850-3000 |
str |
CH3, CH2 & CH |
1350-1470 |
med |
CH2 & CH3
deformation |
|
3020-3100 |
med |
=C-H & =CH2 (usually
sharp) |
880-995 |
str |
=C-H & =CH2 |
|
|
Alkynes |
3300 |
str |
C-H (usually sharp) |
600-700 |
str |
C-H deformation |
|
3030 |
var |
C-H (may be several bands) |
690-900 |
str-med |
C-H bending & |
|
|
3580-3650 |
var |
O-H (free), usually sharp |
1330-1430 |
med |
O-H bending (in-plane) |
|
|
3400-3500 (dil. soln.) |
wk |
N-H (1°-amines), 2 bands |
1550-1650 |
med-str |
NH2 scissoring (1°-amines) |
|
|
2690-2840(2
bands) 1690 1675 1745 1780 |
med |
C-H (aldehyde C-H) |
|
|
|
|
|
2500-3300
(acids) overlap C-H 1785-1815 ( acyl
halides) 1750 &
1820 (anhydrides)
1040-1100 1735-1750
(esters)
1000-1300 1630-1695(amides) |
str |
O-H (very broad) |
1395-1440 |
med |
C-O-H bending |
|
|
Nitriles |
2240-2260 |
med |
C≡N (sharp) |
|
||
NUCLEAR
MAGNETIC RESONANCE SPECTROSCOPY
Nuclear magnetic resonance spectroscopy, most commonly known as NMR
spectroscopy, is a research technique that exploits the magnetic properties
of certain atomic nuclei. It determines the physical and chemical properties of
atoms or the molecules in which they are contained. It relies on the phenomenon
of nuclear magnetic resonance and can provide detailed information about the
structure, dynamics, reaction state, and chemical environment of molecules.
When
placed in a magnetic field, NMR active nuclei (such as 1H or 13C)
absorb electromagnetic radiation at a frequency characteristic of the isotope.
The resonant frequency, energy of the absorption, and the intensity of
the signal are proportional to the strength of the magnetic field.
Nuclear spin and the splitting of energy levels in
a magnetic field
Subatomic
particles (electrons, protons and neutrons) can be imagined as spinning on
their axes. In many atoms (such as 12C) these spins are paired
against each other, such that the nucleus of the atom has no overall spin.
However, in some atoms (such as 1H and 13C) the nucleus
does possess an overall spin. The rules for determining the net spin of a
nucleus are as follows;
- If
the number of neutrons and the number of protons are both even,
then the nucleus has NO spin.
- If
the number of neutrons plus the number of protons is odd, then the
nucleus has a half-integer spin (i.e. 1/2, 3/2, 5/2)
- If
the number of neutrons and the number of protons are both odd, then
the nucleus has an integer spin (i.e. 1, 2, 3)
Chemical shift
The
magnetic field at the nucleus is not equal to the applied magnetic
field; electrons around the nucleus shield it from the applied field. The
difference between the applied magnetic field and the field at the nucleus is
termed the nuclear shielding.
Consider
the s-electrons in a molecule. They have spherical symmetry and circulate in
the applied field, producing a magnetic field which opposes the applied field.
This means that the applied field strength must be increased for the nucleus to
absorb at its transition frequency. This upfield shift is also termed diamagnetic
shift.
Electrons
in p-orbitals have no spherical symmetry. They produce comparatively
large magnetic fields at the nucleus, which give a low field shift. This
"deshielding" is termed paramagnetic shift.
Spin -
spin coupling
Consider
the structure of ethanol;
The 1H
NMR spectrum of ethanol (below) shows the methyl peak has been split into three
peaks (a triplet) and the methylene peak has been split into four peaks
(a quartet). This occurs because there is a small interaction (coupling)
between the two groups of protons. The spacing between the peaks of the methyl
triplet are equal to the spacings between the peaks of the methylene quartet.
This spacing is measured in Hertz and is called the coupling constant, J.
To see why the methyl peak is split
into a triplet, let's look at the methylene protons. There are two of
them, and each can have one of two possible orientations (aligned with or
opposed against the applied field). This gives a total of four possible states;
In the first possible combination,
spins are paired and opposed to the field. This has the effect of reducing the
field experienced by the methyl protons; therefore a slightly higher
field is needed to bring them to resonance, resulting in an upfield shift.
Neither combination of spins opposed to each other has an effect on the methyl
peak. The spins paired in the direction of the field produce a downfield shift.
Hence, the methyl peak is split into three, with the ratio of areas 1:2:1.
Similarly, the effect of the methyl
protons on the methylene protons is such that there are eight possible spin
combinations for the three methyl protons;
Out of these eight groups, there are
two groups of three magnetically equivalent combinations. The methylene peak is
split into a quartet. The areas of the peaks in the quartet have the ration
1:3:3:1.
STRUCTURAL ELUCIDATION USING NMR SPECTROSCOPY
The note is that structure system is
A3M2X2. Ha and Hx has
the triplet pattern by Hm because of N+1 rule. The signal of Hm is split into
six peaks by Hx and Ha.
1) the structure of 2-methyl propanal is drawn
2) Figure out which protons are chemically equivalent, i.e.,
two methyl (-CH3) groups are chemical equivalent.
3) Chemical
shift of each protons is predicted by 1H chemical shift ranges
(Fig.1): chemical shift of methyl groups (Ha) : 1-2 ppm (?Ha=1.1
ppm); chemical shift of -CH- groups (Hb) moves to downfield due to
effect on aldehyde groups:2-3ppm ( ?Hb=2.4 ppm); chemical shift of
aldehyde groups (Hc):9-10 ppm (?Hc=9.6 ppm)
4)
Splitting pattern is determined by (N+1) rule: Ha is split into two peaks by Hb(#of
proton=1). Hb has the septet pattern by Ha (#of
proton=6). Hc has one peak. (Note that Hc has
doublet pattern by Hb due to vicinal proton-proton coupling.)
MASS
SPECTROMETRY
Mass
spectrometry (MS) is an analytical technique that produces
spectra (singular spectrum) of the masses of the molecules comprising a
sample of material. The spectra are used to determine the elemental composition
of a sample, the masses of particles and of molecules, and to elucidate the
chemical structures of molecules, such as peptides and other chemical compounds.
Mass spectrometry works by ionizing chemical compounds to generate charged
molecules or molecule fragments and measuring their mass-to-charge ratios.
In a
typical MS procedure, a sample, which may be solid, liquid, or gas, is ionized.
The ions are separated according to their mass-to-charge ratio The ions are
detected by a mechanism capable of detecting charged particles. Signal
processing results are displayed as spectra of the relative abundance of ions
as a function of the mass-to-charge ratio. The atoms or molecules can be
identified by correlating known masses to the identified masses or through a
characteristic fragmentation pattern.
INSTRUMENTATION:
A mass
spectrometer consists of three components: an ion source, a mass analyzer, and
a detector. The ionizer converts a portion of the sample into ions.
There are a wide variety of ionization techniques, depending on the phase
(solid, liquid, gas) of the sample and the efficiency of various ionization
mechanisms for the unknown species. An extraction system removes ions from the
sample, which are then trajected through the mass analyzer and onto the detector.
The differences in masses of the fragments allows the mass analyzer to sort the
ions by their mass-to-charge ratio. The detector measures the value of an
indicator quantity and thus provides data for calculating the abundances of
each ion present.
Alcohol
An alcohol's molecular ion is small
or non-existent. Cleavage of the C-C bond next to the oxygen usually
occurs. A loss of H2O may occur as in the spectra below.
3-Pentanol (C5H12O)
with MW = 88.15
|
|
Aldehyde
Cleavage of bonds next to the
carboxyl group results in the loss of hydrogen (molecular ion less 1) or the
loss of CHO (molecular ion less 29).
3-Phenyl-2-propenal (C9H8O)
with MW = 132.16
Alkane
Molecular
ion peaks are present, possibly with low intensity. The fragmentation
pattern contains clusters of peaks 14 mass units apart (which represent loss of
(CH2)nCH3).
Hexane
(C6H14) with MW = 86.18
Measurement of Ph of different samples using Ph meter.
EQUIPMENT:
Ph meter
SAMPLE:
Hydrochloric acid
Nitric acid
Sodium hydroxide
Water
PROCEDURE:
·
First
of all make sure that pH meter is clean and make it in working condition
·
Take
the sample whose pH is to be determined in a beaker
·
Dip Ph
meter, measure for few seconds i.e for 3.5 sec
·
Required
measurement will be obtained
·
Repeat
same procedure for other compounds and measure their Ph values.
·
Record
values in an table
|
S.NO |
PRACTICALS |
|
1. |
SEPARATION OF MIXTURE OF
INK USING PAPER CHROMATOGRAPHY |
|
2. |
SEPARATION OF MIXTURE OF SUBSTANCE USING 2-D
CHROMATOGRAPHY
|
|
3. |
SEPARATION OF
MIXTURE USING CIRCULAR PAPER CHROMATOGRAPHY
|
|
4. |
COMPARISON OF RF
VALUE OF ASPIRIN AND SALICYLIC ACID USING TLC.
|
|
5. |
SEPARATION OF BETA CAROTENE AND CHLOROPHYLL FROM SPINACH LEAVES BY
COLUMN CHROMATOGRAPHY
|
|
6. |
DETERMINATION OF
VISCOSITY OF DIFFERENT PHARMACEUTICAL FLUIDS.
|
|
7. |
USE OF POLARIMETER AND MEASUREMENT OF ANGLE OF ROTATION OF AN
OPTICALLY ACTIVE SUBSTANCE.
|
CHROMATOGRAPHY
Chromatography is a physical method of
separation that distributes components to separate between two phases, one
stationary (stationary phase), the other (the mobile phase) moving in a
definite direction.
Chromatography
may be preparative or analytical. The purpose of preparative chromatography is
to separate the components of a mixture for more advanced use (and is thus a
form of purification). Analytical chromatography is done normally with smaller
amounts of material and is for measuring the relative proportions of analytes
in a mixture.
Chromatography terms
·
The analyte is the substance to be
separated during chromatography.
·
Analytical
chromatography is used to determine the existence and possibly also the
concentration of analyte(s) in a sample.
·
A bonded phase is a stationary phase
that is covalently bonded to the support particles or to the inside wall of the
column tubing.
·
A chromatogram is the visual output of
the chromatograph. In the case of an optimal separation, different peaks or
patterns on the chromatogram correspond to different components of the
separated mixture.
·
A chromatograph
is equipment that enables a sophisticated separation, e.g. gas chromatographic
or liquid chromatographic separation.
·
The eluate is the mobile phase leaving
the column.
·
The eluent is the solvent that carries
the analyte.
·
An eluotropic
series is a list of solvents ranked according to their eluting power.
·
An immobilized phase is a stationary
phase that is immobilized on the support particles, or on the inner wall of the
column tubing.
·
The mobile phase is the phase that moves
in a definite direction. It may be a liquid (LC and Capillary
Electrochromatography (CEC)), a gas (GC), or a supercritical fluid
(supercritical-fluid chromatography, SFC). The mobile phase consists of the
sample being separated/analyzed and the solvent that moves the sample through
the column. In the case of HPLC the mobile phase consists of a non-polar
solvent(s) such as hexane in normal phase or polar solvents in reverse phase
chromotagraphy and the sample being separated. The mobile phase moves through
the chromatography column (the stationary phase) where the sample interacts
with the stationary phase and is separated.
·
Preparative chromatography is used to purify sufficient
quantities of a substance for further use, rather than analysis
·
The retention time is the characteristic
time it takes for a particular analyte to pass through the system (from the
column inlet to the detector) under set conditions.
·
The sample
is the matter analyzed in chromatography. It may consist of a single component
or it may be a mixture of components. When the sample is treated in the course
of an analysis, the phase or the phases containing the analytes of interest
is/are referred to as the sample whereas everything out of interest separated
from the sample before or in the course of the analysis is referred to as
waste.
·
The solute
refers to the sample components in partition chromatography.
·
The solvent refers to any substance
capable of solubilizing another substance, and especially the liquid mobile phase
in liquid chromatography.
·
The stationary phase is the substance
fixed in place for the chromatography procedure. Examples include the silica
layer in thin layer chromatography
·
The detector
refers to the instrument used for qualitative and quantitative detection of
analytes after separation.
Techniques by chromatographic bed shape
Column chromatography
Column
chromatography is a separation technique in which the stationary bed is within
a tube. The particles of the solid stationary phase or the support coated with
a liquid stationary phase may fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside tube wall leaving an open,
unrestricted path for the mobile phase in the middle part of the tube (open tubular
column). Differences in rates of movement through the medium are calculated to
different retention times of the sample.
Planar chromatography
Planar
chromatography is a separation technique in which the stationary phase is
present as or on a plane. The plane can be a paper, serving as such or
impregnated by a substance as the stationary bed (paper chromatography) or a
layer of solid particles spread on a support such as a glass plate (thin layer
chromatography). Different compounds in the sample mixture travel different
distances according to how strongly they interact with the stationary phase as
compared to the mobile phase. The specific Retention factor (Rf) of
each chemical can be used to aid in the identification of an unknown substance.
Paper chromatography
Paper chromatography is a
technique that involves placing a small dot or line of sample solution onto a
strip of chromatography paper. The paper is placed in a jar containing a
shallow layer of solvent and sealed. As the solvent rises through the paper, it
meets the sample mixture, which starts to travel up the paper with the solvent.
This paper is made of cellulose, a polar substance, and the compounds within
the mixture travel farther if they are non-polar. More polar substances bond with
the cellulose paper more quickly, and therefore do not travel as far.
Thin layer chromatography
Thin
layer chromatography (TLC) is a widely employed laboratory technique and is
similar to paper chromatography. However, instead of using a stationary phase
of paper, it involves a stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose on a flat, inert substrate. Compared to
paper, it has the advantage of faster runs, better separations, and the choice
between different adsorbents. For even better resolution and to allow for
quantification, high-performance TLC can be used.
Techniques by physical state
of mobile phase
Gas chromatography
Gas chromatography is based
on a partition equilibrium of analyte between a solid or viscous liquid
stationary phase (often a liquid silicone-based material) and a mobile gas
(most often helium). The stationary phase is adhered to the inside of a
small-diameter (commonly 0.53 - 0.18mm inside diameter) glass or fused-silica
tube (a capillary column) or a solid matrix inside a larger metal tube (a
packed column). It is widely used in analytical chemistry; though the high
temperatures used in GC make it unsuitable for high molecular weight
biopolymers or proteins (heat denatures them), frequently encountered in
biochemistry, it is well suited for use in the petrochemical, environmental
monitoring and remediation, and industrial chemical fields. It is also used
extensively in chemistry research.
Liquid chromatography
Liquid
chromatography (LC) is a separation technique in which the mobile phase is a
liquid. Liquid chromatography can be carried out either in a column or a plane.
Present day liquid chromatography that generally utilizes very small packing
particles and a relatively high pressure is referred to as high performance
liquid chromatography (HPLC).
Affinity chromatography:
Affinity chromatography is based on selective non-covalent interaction
between an analyte and specific molecules. It is very specific, but not very
robust. It is often used in biochemistry in the purification of proteins bound
to tags. These fusion proteins are labeled with compounds such as His-tags,
biotin or antigens, which bind to the stationary phase specifically. After
purification, some of these tags are usually removed and the pure protein is
obtained.
Special techniques:
Reversed-phase chromatography:
Reversed-phase
chromatography (RPC) is any liquid chromatography procedure in which the mobile
phase is significantly more polar than the stationary phase. It is so named
because in normal-phase liquid chromatography, the mobile phase is
significantly less polar than the stationary phase. Hydrophobic molecules in
the mobile phase tend to adsorb to the relatively hydrophobic stationary phase.
Hydrophilic molecules in the mobile phase will tend to elute first. Separating
columns typically comprise a C8 or C18 carbon-chain bonded to a silica particle
substrate.
Two-dimensional chromatography:
In
some cases, the chemistry within a given column can be insufficient to separate
some analytes. It is possible to direct a series of unresolved peaks onto a
second column with different physico-chemical (Chemical classification)
properties. Since the mechanism of retention on this new solid support is
different from the first dimensional separation, it can be possible to separate
compounds that are indistinguishable by one-dimensional chromatography. The
sample is spotted at one corner of a square plate,developed, air-dried, then
rotated by 90° and usually redeveloped in a second solvent system.
Paper chromatography
Paper chromatography is an analytical method
technique for separating and identifying mixtures that are or can be coloured,
especially pigments. This can also be used in secondary or primary colours in
ink experiments. This method has been largely replaced by thin layer
chromatography, however it is still a powerful teaching tool.
Double-way paper
chromatography, also called two-dimensional chromatography,
involves using two solvents and rotating the paper 90° in between. This is
useful for separating complex mixtures of similar compounds, for example, amino
acids. if a filter paper is used, it should be of a high quality paper. The
mobile phase is developing solutions that can travel up to the stationary phase
carrying the sample alongside with it.
Types of Paper Chromatography
1.Descending
Paper Chromatography-In this type development of chromatogram is done by
allowing the solvent to travel down the paper is called Descending
Chromatography. Here the mobile phase is present in the upper portion.
2.Ascending
Paper Chromatography-Here the solvent travel upward direction of the
Chromatographic paper. Both the Descending and Ascending Paper Chromatography
are used for separation of Organic and Inorganic substances.
3.Ascending-Descending
Paper Chromatography-It is the hybrid of both the above technique. The upper
part of the Ascending chromatography can be folded over a rod and allowing the
paper to become descending after crossing the rod.
4.Radial
Paper Chromatography-It is also called as Circular chromatography. Here a
circular filter paper is taken and the sample is given at the center of the
paper. After drying the spot the filter paper tied horizontally on a Petridish
containing solvent. So that Wick of the paper is dipped inside the solvent. The
solvent rises through the wick and the component get separated in form of
concentrate circular zone.
5.Two-Dimensional
Paper Chromatography-In this technique a square or rectangular paper is used.
Here the sample is applied to one of the corner and development is performed at
right angle to the direction of first run.
RETENTION FACTOR:
The
retention factor (Rƒ) may be defined as the ratio of the
distance traveled by the substance to the distance traveled by the solvent. If
Rƒ value of a solution is zero, the solute remains in the
stationary phase and thus it is immobile. If Rƒ value = 1
then the solute has no affinity for the stationary phase and travels with the
solvent front. To calculate the Rƒ value, take the distance traveled
by the substance divided by the distance traveled by the solvent. For example,
if a compound travels 2.1 cm and the solvent front travels 2.8 cm, (2.1/2.8)
the Rƒ value = 0.75
SEPARATION OF MIXTURE OF INK USING PAPER
CHROMATOGRAPHY
Apparatus:
Whatmann no.1
filter paper, chromatographic tank, capillary tube, pencil
Chemicals:
Mixture of
inks, red, blue, black and yellow ink
Procedure:
·
The spot of the test sample is loaded on the filter
paper using a capillary tube. This spot should always be a concentrated but a
very minute one.
·
Capillary tubes are used in paper chromatography,
because a small quantity can be taken into the tube without any force.
·
The upper line (solvent front) is drawn on the paper
from 2 cm on the top and the bottom line (base line) is drawn from 2 cm from
the bottom of the paper.
·
Usually 0.01g of the sample is dissolved in the
running solvent (1g). Micro liter quantities are used to spot on the paper by a
capillary tube.
·
The diameter of the spot should be only up to few mili
meters. The spotting should be done several times in order to get a
concentrated spot.
·
This filter paper is then placed inside the chamber
saturated with the solvent to develop the chromatogram.
·
The chromatogram is then heated in an oven to high
temperatures. This can be done not only by an oven but also using a fan, air
etc.
SEPARATION OF MIXTURE OF SUBSTANCE USING 2-D CHROMATOGRAPHY
Double-way paper chromatography, also called two-dimensional
chromatography, involves using two solvents and rotating the paper 90° in
between. This is useful for separating complex mixtures of similar compounds,
for example, amino acids. if a filter paper is used, it should be of a high
quality paper. The mobile phase is developing solutions that can travel up to
the stationary phase carrying the sample alongside with it.
Two way paper chromatography gets around the problem of separating
out substances which have very similar Rf values.
PROCEDURE:
·
Cut out a 4.5̋̋̋̋̋̋ x 4.5cm filter paper.
·
Draw a line on filter paper using a pencil about 2
cm from a baseline.
·
Apply small spots of mixture of substances and
individual components to the pencil line.
·
Place the filter paper in the chromatographic tank.
·
Allow the chromatogram to develop until solvent front
reaches around 1cm of the top.
·
Dry the chromatogram and turn out 90 ̊C in another beaker.
·
The component of mixture will separate into different
components further owing to their presence for various components.
·
Calculate their Rf value.
SEPARATION
OF MIXTURE USING CIRCULAR PAPER CHROMATOGRAPHY
Radial
chromatography is a form of paper chromatography, a technique for
separating colored substances from their mixtures.
Here
the solvent travels from the center of the circular chromatography paper
towards the periphery. The entire system is kept in a covered petri dish in
order to develop a chromatogram.
The
wick at the center of paper dips into mobile phase in a petridish by which the
solvent drains on to the paper and moves the sample radially to form the sample
spots of different compounds as concentric rings.
PROCEDURE:
·
Apply
a small amount of mixture to be separated to the center of circular filter
paper.
·
Dip
the center of filter paper into petri dish and then cover it.
·
Allow
the chromatogram to develop until the solvent front reaches about 1 inch short
of paper border.
·
Remove
the chromatogram from the petridish and allow it to air dry.
·
Calculate
Rf value of each separated component.
THIN LAYER CHROMATOGRAPHY
Thin layer chromatography (TLC) is a chromatography
technique used to separate non-volatile mixtures. Thin layer chromatography
is performed on a sheet of glass, plastic, or aluminium foil, which is coated
with a thin layer of adsorbent material, usually silica gel, aluminium oxide,
or cellulose. This layer of adsorbent is known as the stationary phase.
After
the sample has been applied on the plate, a solvent or solvent mixture (known
as the mobile phase) is drawn up the plate via capillary action. Because
different analytes ascend the TLC plate at different rates, separation is
achieved.
Thin
layer chromatography can be used to monitor the progress of a reaction,
identify compounds present in a given mixture, and determine the purity of a
substance.
PRINCIPLE:
Similar
to other chromatographic methods TLC is also based on the principle of
separation. The separation depends on the relative affinity of compounds
towards stationary and mobile phase. The compounds under the influence of
mobile phase (driven by capillary action) travel over the surface of stationary
phase. During this movement the compounds with higher affinity to stationary
phase travel slowly while the others travel faster. Thus separation of
components in the mixture is achieved.
PLATE PREPARATION:
TLC
plates are usually commercially available, with standard particle size ranges
to improve reproducibility. They are prepared by mixing the adsorbent, such as
silica gel, with a small amount of inert binder like calcium sulfate (gypsum)
and water. This mixture is spread as thick slurry on an un reactive carrier
sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is
dried and activated by heating in an oven for thirty minutes at
110 °C. The thickness of the absorbent layer is typically around 0.1 –
0.25 mm for analytical purposes and around 0.5 – 2.0 mm for
preparative TLC.
COMPARISON OF RF VALUE OF
ASPIRIN AND SALICYLIC ACID USING TLC.
CHEMICALS:
Aspirin and salicylic acid
MOBILE
PHASE:
Acetone:
ether (1:1)
procedure:
A thin mark
is made at the bottom of the plate with a pencil to apply the sample spots.
Then samples
solutions are applied on the spots marked on the line at equal
distances.
The mobile phase is poured into the TLC chamber to a level
few centimeters above the chamber bottom
Then
the plate prepared with sample
spotting is placed in TLC chamber such that the side of the plate with sample
line is towards the mobile phase. Then the chamber is closed with a
lid.
The plate is
immersed such that sample spots are well above the level of mobile phase but
not immersed in the solvent as shown in the picture for development.
Allow sufficient time for development of spots.
Then the plates are removed and allowed to dry. The sample spots are visualized
in suitable UV light chamber .
QUESTION:
Which one will migrate more with non polar
mobile phase? Why? Justify your answer.
Advantages of TLC
The Thin layer chromatography advantages include:
It
is simple process with short development time.
It
helps in visualization of separated compound spots easily.
The
method helps to identify the individual compounds.
It
helps in isolation of most of the compounds.
The
separation process is faster and the selectivity for compounds is higher (even
small differences in chemistry is enough for clear separation.
The
purity standards of the given sample can be assessed easily.
It
is a cheaper chromatographic technique
COLUMN CHROMATOGRAPHY
Column chromatography is a method used to purify
individual chemical compounds from mixtures of compounds. It is often used for
preparative applications on scales from micrograms up to kilograms. The main
advantage of column chromatography is the relatively low cost and disposability
of the stationary phase used in the process. The latter prevents
cross-contamination and stationary phase degradation due to recycling.
The
classical preparative chromatography column, is a glass tube with a diameter
from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and
some kind of a filter (a glass frit or glass wool plug – to prevent the loss of
the stationary phase) at the bottom. Two methods are generally used to prepare
a column: the dry method, and the wet method.
·
For
the dry method, the column is first filled with dry stationary phase
powder, followed by the addition of mobile phase, which is flushed through the
column until it is completely wet, and from this point is never allowed to run
dry.
·
For
the wet method, a slurry is prepared of the eluent with the stationary
phase powder and then carefully poured into the column. Care must be taken to
avoid air bubbles. A solution of the organic material is pipetted on top of the
stationary phase. This layer is usually topped with a small layer of sand or
with cotton or glass wool to protect the shape of the organic layer from the
velocity of newly added eluent. Eluent is slowly passed through the column to
advance the organic material. Often a spherical eluent reservoir or an
eluent-filled and stoppered separating funnel is put on top of the column.
SEPARATION
OF BETA CAROTENE AND CHLOROPHYLL FROM SPINACH
LEAVES BY COLUMN CHROMATOGRAPHY
Introduction
In this
experiment, you will use column chromatography to separate and isolate two
colored compounds
from spinach leaves. The first is beta carotene, which is the precursor to
vitamin A, and has a yellow color. The second is chlorophyll, which plants use
in the process of photosynthesis, and has a green color.
Column
chromatography operates on the same principles as TLC, but instead of using a
plate coated with silica gel, you will use a buret filled with alumina. Solvent
will travel down the column instead of up a plate. The same concepts of
polarity apply.
Beta
carotene is a hydrocarbon, making it quite nonpolar. Chlorophyll contains very
polar
bonds to magnesium as well as a few polar functional groups. This large
difference in polarity makes this separation very effective. The beta carotene
moves much more easily down the column than the chlorophyll.
Both beta
carotene and chlorophyll are colored, which will make it easy to observe their
movement down the column. The color comes from light being absorbed by the
compounds because of their numerous C=C bonds.
Procedure
Separate the beta carotene and chlorophyll from the
spinach leaves:
• Obtain
10-14 spinach leaves. Pinch off the stems and wash off any dirt. Pat them as
dry
as you can with paper towels. Then add leaves to your
pile until you have at least 10 g of
leaves.
• Place the
leaves in a large mortar and add about 20 ml of dichloromethane and 20 ml of
petroleum ether. Mush the leaves carefully with a
pestle for about 5 minutes to extract all
organic-soluble components.
• Carefully
pour off the organic solvents into a 100 or 50 ml round bottom flask, leaving any
water (which will be at the bottom) behind.
• Add about
500 mg of alumina to the flask. Rotovap to dryness, as described in
“Evaporating
a Solution.”
Separate
the beta carotene and chlorophyll from each other:
•
Prepare a column as described in
"Separating Compounds by Column
Chromatography."
·
Pour the
sample plus alumina in the top of the column and wash it down with a few ml of
hexanes if it sticks to the sides.
·
Prepare a
solution of 10% ethyl acetate in hexanes (start with 100 ml and
prepare more as
needed).
·
Fill the
column with solvent, open the stopcock, and begin pushing it through with the
bulb. At this point a yellow band should appear and move down the column – this
is the beta carotene.
·
Collect
it in a separate flask as it comes out the bottom (start
collecting
when the yellow band is about an inch from the
bottom)
when the yellow band has been completely
eluted
push
out any extra solvent.
• Prepare 100 ml of 10% methanol in dichloromethane.
·
Add the
new solvent to the column and begin pushing it through a green band should now move down the column.
• Collect this band in a separate flask as it is eluted from the column.
• Force all liquid from the column and leave it on my desk. (It is
very
difficult to clean it out until it dries.)
QUESTIONS:
·
Which is
more polar, beta carotene or chlorophyll? If you didn’t have access to their structures,
how would you know this from the experiment?
·
Which
solvent system is more polar, 10% ethyl acetate in
hexanes, or 10% methanol in
dichloromethane? How can you
tell from the experiment?
VISCOSITY
viscosity is the quantity that describes a
fluid's resistance to flow. Fluids resist the relative motion of immersed
objects through them as well as to the motion of layers with differing
velocities within them.
Viscosity
is the measure of the internal friction of a fluid. This friction becomes apparent
when a layer of fluid is made to move in relation to another layer. The greater
the friction, the greater the amount of force required to cause this movement,
which is called shear. Shearing occurs whenever the fluid is
physically moved or distributed, as in pouring, spreading, spraying, mixing,
etc. Highly viscous fluids, therefore, require more force to move than less
viscous materials.
DETERMINATION
OF VISCOSITY OF DIFFERENT PHARMACEUTICAL FLUIDS.
BROOK FIELD VISCOMETER:
The
rotational viscometer is based upon the measurement of torque of a rotating
spindle a sample at a specified velocity.
The
instrument based on the fact that solid rotating body immersed in a liquid is
subjected to the retarding force due to the viscous drag.
The
force is directly proportional to the viscosity of the substance.
Apparatus:
Brook
field viscometer
Chemicals:
Glycerol,
Chloroform, acetone, ether, 70℅ sucrose.
Procedure:
Adjust
the bubble level at the top of the instrument.
Introduce
the medicinal compound in a beaker.
Place
spindle of suitable size.
Adjust
the spindle number and the speed required.
Turn
on the viscometer and note the viscosity.
Repeat
the procedure on all the fluids.
The
obtained values can be compared with literature values to determine the viscosity
of a substance.
USE OF
POLARIMETER AND MEASUREMENT OF ANGLE OF ROTATION OF AN OPTICALLY ACTIVE
SUBSTANCE.
A polarimeter is a scientific instrument used to
measure the angle of rotation caused by passing polarized light through an optically active substance.
Some chemical substances are optically active, and polarized
(unidirectional) light will rotate either to the left (counter-clockwise) or
right (clockwise) when passed through these substances. The amount by which the
light is rotated is known as the angle of rotation.
Construction
The polarimeter is made up of two Nicol prisms (the polarizer and analyzer). The polarizer is fixed and the analyzer can be
rotated. The prisms may be compared to as slits S1 and S2. The light waves may
be considered to correspond to waves in the string. The polarizer S1 allows
only those light waves which move in a single plane. This causes the light to
become plane polarized. When the analyzer is also placed in a similar position
it allows the light waves coming from the polarizer to pass through it. When it
is rotated through the right angle no waves can pass through the right angle
and the field appears to be dark. If now a glass tube containing an optically
active solution is placed between the polarizer and analyzer the light now
rotates through the plane of polarization through a certain angle, the analyzer
will have to be rotated in same angle.
Operation
Polarimeters measure this by passing monochromatic light through the first of two polarising
plates, creating a polarized beam. This first plate is known as the polarizer. This beam is then rotated as it passes
through the sample. The sample is usually prepared as a tube where the
optically active substance is dissolved in an optically inactive chemical such
as distilled water, ethanol, methanol. Some polarimeters can be
fitted with tubes that allow for sample to flow through continuously.
After passing through the sample, a second polarizer, known
as the analyzer, rotates either via manual rotation or automatic detection of
the angle. When the analyzer is rotated to the proper angle, the maximum amount
of light will pass through and shine onto a detector.
Apparatus:
Polari meter, glass tube.
Chemicals:
D-Glucose.
Procedure:
Turn on
polarimeter and allow to heat for 5min
Make a 1℅
w/v D-Glucose solution and fill in the 100mm test tube provided along with the polarimeter.
Check for
zero position of dial. Incase of an error, add or subtract error value during
procedure.
Ideally
at zero position viewing field should show a darker central region against a
bright region.
Once
viewing field is adjusted, open lens cover and put the test tube containing
glucose solution in it.
Close the
cover with bulb part of tube upward in order to store air bubbles in it.
Throughout
the observation process adjust the dial such that viewing field become
identical.
Read out
angle of light rotation. Calculate specific rotation of unknown compound.
If all other parameters are known. The
equipment can also be utilized to determine concentration of unknown substance
specific rotation can be found by formula:
In this equation, l is
the path length in decimeters and c is the concentration in g/mL, for a
sample at a temperature T (given in degrees Celsius) and wavelength λ
(in nanometers). If the wavelength of the light used is 589 nanometer (the
sodium D line), the symbol “D” is used. The sign of the rotation (+ or −) is
always given. When using this equation, the concentration and the solvent may
be provided in parentheses after the rotation. The rotation is reported using
degrees, and no units of concentration are given (it is assumed to be g/100mL).
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