Chromatographic Methods

Introduction:
Chromatography is one of the accurate techniques used for analytical separations. This
method was invented by a Russian botanist Mikhael Tswett for separating plant pigments. The
term chromatography is from Greek word chorma means 'colour' and graphein means 'to write
No other separation method is as power as chromatography
Principle:
All chromatographic techniques have two immiscible phases:
1. Stationary Phase: This is a fixed phase. Eg: a column of adsorbent, paper, a thin film of liquid
supported on inert solid, a thin layer of adsorbent coated over a glass plate etc.
2. Mobile Phase: This is a moving phase which can be a liquid or gas. The components to be
analyzed are carried by this mobile phase through the stationary phase.
The components of the mixture are separated depending upon the rates at which they are carried
by the mobile phase through the stationary phase, which in turn depends on the relative affinity
of the components towards the stationary phase and mobile phase.

Classification
1. Based on mechanism of separation, chromatography can be classified as:
(a) Adsorption Chromatography: In this type the stationary phase is a solid and the mobile phase
is a liquid or gas. The separation occurs due to difference in adsorption coefficient of the
components.
b) Partition chromatography: In this chromatography stationary phase is a liquid supported on
inert solid and mobile phase is a liquid or gas. Here separation occurs due to the difference in the
partition coefficients of the components.
c) Ion exchange chromatography: In this chromatography stationary phase is an ion exchanger
and the separation of the mixture is based on ion exchange principle and applicable for ionic
species.
Classification of chromatography based on the mobile phase,
a) Liquid chromatography:
If the mobile phase is liquid and the stationary phase is solid, then the chromatography is called
LSC (Liquid Solid Chromatography) If the mobile phase is liquid and the stationary phase is liquid
supported on solid, then the chromatography is called LLC (Liquid Liquid Chromatography)
b) Gas chromatography:
If the mobile phase is gas and the stationary phase is solid, then the chromatography is called
GSC (Gas Solid Chromatography). If the mobile phase is gas and the stationary phase is liquid,
then the chromatography is called GLC (Gas Liquid Chromatography)

Column Chromatography
Principle:

 The components of a mixture are separated depending upon extent of adsorption on the
stationary phase and the rate at which component is carried by the mobile phase.
 Column chromatography is one of the most useful methods for the separation and
purification of both solids and liquids.
 This is a solid liquid technique in which the stationary phase is a solid and mobile phase is
a liquid.
 The usual adsorbents employed column chromatography are silica, alumina, calcium
carbonate, calcium phosphate, magnesia, starch, etc., selection of solvent is based on the
nature of both the solvent and the adsorbent.
 The rate at which the components of a mixture are separated depends on the activity or
polarity of the adsorbent and polarity of the solvent.
o If activity of the adsorbent is very high and polarity of the solvent is very low, then
the separation is very slow but gives a good separation. On the other hand,
o if the activity of adsorbent is low and polarity of the solvent is high the separation
is rapid but gives only a poor separation,
o ie., the components separated are not 100 percentage pure.
o For example, when silica gel (an adsorbent with high polarity) is used as adsorbent
we should start eluting with a solvent like hexane (low polarity) for good
separation.
Procedure:
 The adsorbent is packed in a cylindrical tube and is plugged at the bottom by a piece of
glass wool or porous disc.
 The mixture to be separated is dissolved in a suitable solvent and introduced at the top
of the column and is allowed to pass through the column.
 As the mixture moves down through the column, the components are adsorbed at
different regions depending on their ability for adsorption.
 The component with greater adsorption power will be adsorbed at the top and the other
will be adsorbed at the bottom.
 The different components can be desorbed and collected separately by adding more
solvent at the top and this process is known as elution. That is, the process of dissolving
 out of the components from the adsorbent is called elution and the solvent is called
eluant.
 The weakly adsorbed component will be eluted more rapidly than the other.
 The different fractions are collected separately.
 The purity of each fraction is monitored using TLC.
 Like fractions are clubbed.
 Distillation or evaporation of the solvent from the different fractions gives the pure
components.

Image: Column chromatography

Applications
1. Column chromatography is best suited to separate organic compounds from plant
materials
2. In separation of compounds after organic synthesis to obtain desired molecule
3. To separate or purify natural compound mixtures like alkaloids, glycosides etc.
4. Column chromatography is used for large scale separation and purification of phar
maceuticals.

Thin layer Chromatography

Principle:

 The components of a mixture are separated depending upon extent adsorption on the
stationary phase and the rate at which component is carried by the mobile phase.
 TLC can be used to determine not only the number of components in a mixture but al
to identify the compounds and the purity of a compound.
 TLC is a sensitive technique microgram quantities can be analyzed by TLC and it takes
little time for analysis.
 Commercially available TLC plate is a sheet of glass, metal, or plastic which is coated
with a thin layer of a solid adsorbent usually silica or alumina.
  TLC plates can also be prepared in the laboratory by using silica gel powder for TLC.
 The slurry of this powder is made in water and spread over glass plates. The coated
plates are dried and activated by heating in an oven at 120°C.
Procedure:
 A small amount of the mixture solution to be analyzed is spotted near the bottom end
of this plate using a capillary tube (about 1 cm above bottom end)
 The TLC plate is then placed in a shallow pool of a solvent in a developing chamber
such that only the bottom end of the plate touches the solvent.
 The lid of the developing chamber is closed. The solvent very slowly rises up the TLC
plate by capillary action.
 As the solvent passes the spot, it carries the components at different rate, resulting in
separation of the compounds.
 When the solvent has reached near the top of the plate, the plate is removed from
the developing chamber, dried and the separated components of the mixture are
visualized.
 Usually the compounds are not coloured,
so the following visualization techniques are used.
1. UV lamp (254 nm) is used to visualize the plates. Organic compounds containing benzene
rings are UV fluorescent and appear as bright spots. Some commercially available plate
itself contains a fluorescent dye which glows everywhere except where an organic
compound is on the plate. Thus different compounds appear as black spot in bright
background.
2. Iodine vapours are also use to identify the components. After developing TLC plate it is
placed in closed jar contains iodine crystals. The sublimed iodine gets deposited on the
plate, the spots of organic compounds appear as brown, since more amounts iodine is
deposited on the organic spots than silica adsorbent surface.
3. There are also specific colour reagents which can be sprayed onto the plates eg:
Potassium permanganate solution, 2 % H₂SO₄ spray, Vanilillin-H2SO4 spray. Ninhydrin
spray, 10 % NaOH spray. After spraying, the plates are heated in an oven to develop
colours.
Retention Factor(R):
After a separation is complete, individual compounds appear as spots separated vertically.
Each spot has a retention factor (R), which is the ratio of distance travelled by the sample
spot to the distance travelled by the solvent front
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑠𝑝𝑜𝑡
Rf =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
The Rf value can be used to identify compounds due to the uniqueness of each compound in a
particular solvent. When comparing two different compounds under the same conditions, the
compound with the larger Rf value is less polar because it does not stick to the stationary phase
silica when compared with a polar compound.
Solvent polarity In column chromatography and TLC polarity of the eluting solvent is very
important.
 The solvent polarity increases in the following order hexane < carbontetrachloride <
benzene < dichloromethane < chloroform < ethylacetate << acetone < ethanol < methanol
< water < aceticucid < formicacid (strongest).
 For the silica coated plates or silica powder filled column, since silica surface being polar
has great affinity towards polar molecules and has affinity towards non polar molecules
thus one has to start eluting with less polar solvents like hexane and can separate no polar
molecules first.
 Then increase the polarity of the eluent as per the above order.
 For alumina coated plates, since alumina surface being non polar, has great
affinity towards non polar molecules, thus one has to start eluting with polar
solvents like formic acid or methanol and decrease the polarity of the eluent as
per the above order

Thin layer chromatography

Applications
1. To check the purity of a sample. For example, purity of drugs can be checked using TLC,
presence of impurities are shown by presence of additional spots.
2. To check adulteration in food. For example, food colourents and preservatives can be identified
by comparing with authentic sample spots.
3. To determine the appropriate solvent for a column-chromatographic separation
4. To monitor a column-chromatographic separation.
5. To monitor the progress of a chemical reaction, by observing the appearance of a product or
the disappearance of a reactant.
Gas Chromatography (GC)
Chemical analysis of a mixtures of volatile organic compounds like hydrocarbon fuels, flavor and
fragrance chemicals are generally done using GC.
Principle:

 The components of a mixture are separated depending upon extent of adsorption or

partition on the stationary phase and the rate at which component is carried by the
mobile phase.
 Here the mobile phase is a gas like nitrogen, argon, helium or hydrogen.
 The stationary phase may be solid or liquid. If the stationary is termed as Gas solid
chromatography and if the stationary phase is a non volatile chromatorgraphy.
 The stationary phase may be solid or liquid.
o If the stationary plains of solid material like silica, alumina or carbon, then the
chromatography in termed as Gas Solid chromatography (GSC)
o if the stationary phase is a nonvolatile liquid held as a thin layer on a solid support, then
the technique is known as Gas Liquid chromatography (GLC).
o When the stationary phase is a solid (GSC) the principle of separation is adsorption, but
when it is a liquid (GLC) the principle of separation is partition.
Instrumentation:
Basically all gas chromatographic instruments, whether GSC or GLC consist of four basic
components
1.Carrier gas: The carrier gas is allowed to flow through the system, carrying the sample in the
vapour state through the column.
o Example for carrier gas He, N2 etc. For selecting a carrier gas, the following considerations
should be taken into account
a) It should be chemically inert
b) It should be suitable for the detector employed and the type of sample analyzed
c) It should give best column performance consistent with required speed of the analysis
2. Sample injection system

 The carrier gas is connected from the gas reservoir to the sample port injector.
 The sample must be converted into vapour state.
 The injection port is heated to a temperature which will ensure rapid vapourisation but
not thermal degradation of the solute.
 The sample is injected by syringe through a silicon rubber diaphragm in the injection port.
 A solid sample may be dissolved in a suitable solvent and injected as solution.
 The solute vapour mixes instantaneously with the flowing carrier gas and is swept in to
the column
3. Columns:
 In the column, the different components in the vapourised samples are separated from
each other by virtue of their difference in interaction with the column packing, the column
is kept inside a programmable furnace.
 Columns are made of stainless steel, copper, nickel or glass. Inner diameters may range
from 1.6 to 9.5 mm. Length is often 3m. Columns are of two types
(a) Capillary column:
 Capillary column is fabricated from capillary tube, the inner surface of
which is coated with a very thin film of the liquid phase.
Eg dimethylpolysiloxane (Methylsilicone: non-polar) and cartowax 20M
(polar phases) are used.
The characteristic features of an ideal liquid stationary phase are low volatility,
thermal stability and chemical inertness.
(b) The packed columns:
 The packed columns are packed with either a solid powder substrate
(GSC). or a liquid coating on an inert solid support (GLC). Materials used as
solid stationary phase should have high surface area and chemical
inertness like finely powdered silica or alumina.
4. Detectors used in GC:
i) Flame Ionization detector (FID)
FID is one of the most widely used detector for GC.
The ionization detectors are based on the electrical conductivity of gases.
Advantages: High sensitivity, low noise, easy to use.
Disadvantage: It causes the destruction of the sample.
ii) Thermal Conductivity Detector (TCD)
It works on the principle that presence of analyte molecule in the gas stream will
produce a change in thermal conductivity.
Advantages: Simple to use, non – destructive in nature and it gives respond to
both organic and inorganic matter.
Disadvantage: Low sensitivity
Some other detectors like thermionic detector (TID), atomic emission detector (AED) and
electron capture detector (ECD) are also used.
 Gas Chromatography

Working:
 In gas chromatography a mixture of vaporized sample and inert carrier gas (mobile phase)
is passed through the stationary phase. Stationary phase is held in a narrow column.
 As the mixture of gases pass through the column, separation of components occurs via
adsorption or partition on the basis of the physical state of the stationary phase.
 In GSC separation of components occurs on the basis of the degree to which they get
adsorbed.
 In GLC, separation of components occurs on the basis of the difference in the partition
coefficients. Since the partition coefficients of individual components in the mixture are
different. So they are carried along the column at different rates.
 The components which leave the column passes leave column passes through the detector
and recorder.
 The detector produces electrical signals and the recorder converts it as a trace on a paper.
 The resultant trace is a plot of signal intensity against time and is called chromatogram.
 By GC even 10-12g quantity of mixtures can be separated and identified. Hence it is an
important analytical technique.
Retention time tR : it is the time taken by the solute to pass between the sample injection port
and the detector. Identification of the component is carried out by the comparison of the GC
retention time against those of the reference standards
Relative peak area: It is the ratio of area of given peak to the total area of all the peaks. The
percentage of each component in a mixture can obtained from relative peak area.
Applications
1. Gas Chromatography is widely used to test the purity of organic compounds. The presence of
impurities are revealed by the presence of additional peaks
2. Gas chromatography coupled with spectroscopic techniques like IR, NMR and mass
spectrometry, helps in the identification of a components mixture of organic compounds.
3. Gas chromatography coupled with mass spectrometry (GC-MS) is widely used for the analysis
of hydrocarbon fuels, perfumes, flavour and fragrance chemicals etc.
4. GC is widely used to study the extent of air pollution.
5. By using GC, the ethyl alcohol content in blood can be determined with high accuracy.
6. Banned drugs used by athletes can the detected by taking GC of blood or urine sample.

High Performance Liquid Chromatography (HPLC)

 HPLC is applied for the separation of organic compounds which are non-volatile in nature.
 Drug molecules or natural product molecules containing large number of carbon atom
such as cholesterol (C27H40O), or in general triterpenoids which contain 30 carbon atoms,
polypeptides etc. can be separated by this technique.
 The compounds which are non-volatile in natures cannot be analyzed using GC and can
be separated by using HPLC.
Principle:

 The components of a mixture are separated depending upon extent of adsorption or

partition on the stationary phase and the rate at which component is carried by the
mobile phase.
 Here the mobile phase is a liquid and the stationary phase can be a solid or liquid. When
the stationary phase is a solid the principle of separation is adsorption and when it is a
liquid the principle of separation is partition.
  The stationary phase can be a solid or liquid, nowadays liquid stationary phase is more in
use.

HPLC
Instrumentation
1. Pump: A programmable pump which can pump solvent from reservoirs and mix them with
definite composition.
2. Pulse damper: It is used to reduce pulses in pumping pressure
3. Guard column A short guard column is kept before the analytical column. This helps to remove
particulate matter present in the solvent and thereby increases the life of analytical column
4. Analytical column They are usually packed with particle in the rouge 3-10µm range. It is found
that the efficiency of HPLC column improves significantly as the particle size is reduced. In order
to achieve reasonable flow rate content pressure pumps
6. Detectors: Detectors used in HPLC are of two types
(a) Bulk property detector: Bulk property detectors spend to bulk proper ties like
refractive indez, dielectric constant, density etc., of the mobile phase When a particular
molecular species are eluted out, these properties will change and detector gives the signal. After
detection, the component is collected in the sampling tube along with solvent. The qualitative
analysis can be done by spectral studio
(b) Solute property detectors: They respond to solute properties such as UV absorbance,
fluorescence properties of the solute molecules which are not possessed by mobile phase for
example paracetamol can be detected using UV. detector since it gives an absorption at 255nm
due to the presence of a benzene ring in the molecule.
Procedure
The liquid mobile phase is pumped at required pressure and flow rate through the
analytical column which contains the stationary phase. The nonvolatile samples are injected to
the analytical column with the help of a micro syringe. The components are carried along the
column at different rates due to the differential distribution between stationary phase and
mobile phase. The elution can be done in two ways
1. Isocratic elution: a single solvent of constant composition is used
2. Gradient elution: two or more solvents that differ very much in polarity in used
The gradient elution is found to be more effective. During elution the ratio of the solvent in varied
in a programmed manner. The gradient elution gives better separation in less time. The common
organic solvents used are n-hexane, benzene, acetone, methanol, etc. The components that
emerge out of the column are detected and recorded.
Applications
1. Used for the separation of non-volatile organic compound
2. Used for the separation of polypeptides
3. More amount of compounds can be separated when compared with GSC and GLC
4. Used in pharmaceutical industries for routine purity checking and separation.
Reference

1. Engineering Chemistry by Dr. Muhammed Arif M, Kavitha P Nair, Dr Annette Fernandez

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