Chapter 1: Chromatography

Chromatography is a technique for separating and identifying the components of a mixture. Many
different forms of chromatography are used but they all work on the same principle. The components of
the mixture have different affinities for two phases: a stationary phase and a mobile phase and so are
separated as the mobile phase moves through the stationary phase. A component which has a strong
attraction for the mobile phase will move quickly, whereas a component with a strong attraction for the
stationary phase will be held back.

If the stationary and mobile phases are carefully chosen, the different components will move at different
speeds and so be separated effectively. Polar compounds, for example, are more likely to move quickly
when the mobile phase is a polar solvent.

Two main types of chromatography

Partition chromatography

Chromatography using a non-volatile liquid stationary phase held on an inert solid surface is known as
partition chromatography. The components distribute themselves between the two phases according to
their relative solubility. Paper chromatography and gas liquid chromatography are examples. The more
soluble or volatile the component, the faster it will move.

Adsorption chromatography

Chromatography which uses a solid stationary phase and a mobile liquid or gas phase is known as
adsorption chromatography. Some components of the mixture are attracted to the solid surface and the
other components which are less strongly bonded travel faster with the mobile phase. Thin-layer
chromatography is an example. As the stationary phase is generally a polar solid, the more polar solutes
are more readily adsorbed than the less polar solutes.

A. Paper chromatography
B. Thin-layer chromatography (TLC)
C. Column chromatography
D. Gas-liquid chromatography (GLC)

Paper chromatography

Paper chromatography uses paper as the stationary phase. The mobile phase used in this experiment is
called "eluting solution" and consists of a mixture containing polar and non polar solvents. The basic
principle is Partition as it is a liquid-liquid interaction. The moisture layer present above the paper
interacts with the mobile phase.

Measuring the Retardation Factor (Rf): Rf = distance spot traveled/distance solvent traveled

The range of Rf values vary from 01.

Thin-layer chromatography (TLC)

Thin-layer chromatography is an example of Adsorption chromatography. It follows the same

procedure as paper chromatography, with small spots of the test solutions placed on the base line using a
capillary tube. The stationary phase is a thin layer of absorbent particles of alumina or silica supported
on a glass or thin plastic plate and the mobile phase is a liquid solvent. The different components
separate and can be identified. The technique is used in qualitative analysis to determine whether a
substance is pure.

Stationary Phase Silica gel G [G stands for gypsum, calcium sulphate]

Column chromatography

Column chromatography in chemistry 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.

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.

Wet method, 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.

Elution of Column may be Isocratic (Composition of mobile phase is remain constants throughout the
process) or Gradient (Composition of mobile phase is varying from time to time normally non-polar to
polar throughout the process)

Gas-liquid chromatography (GLC)

GLC is used to separate and identify small samples of gases and volatile liquids [Volatility and Thermo
stable are two basic requirement of the sample to be analyzed by the GC]. In this technique, the sample
to be analyzed is injected through a self-sealing cap into an oven where it is vaporized. The vapor is then
carried by an unreactive gas, the mobile phase, over a non-volatile liquid stationary phase. Nitrogen and
helium are typical carrier gases; long chain alkanes of high boiling point supported on a silicon dioxide
surface act as the stationary phase.

The components of the sample are partitioned between the stationary and mobile phases, depending on
their relative boiling points and relative solubility in the two phases. They pass through the column at
different rates, each component leaving after a characteristic interval known as the retention time.
Volatile components with low solubility in the liquid stationary phase emerge first from the column (they
have shorter retention times). An unknown compound can be identified by comparing the retention time
with that of known compounds measured under the same conditions. The area under each peak is a
measure of the amount of each substance present. It is important that the temperature of the oven is
carefully controlled as it affects the rate at which molecules move through the apparatus. Methanol has
the lowest boiling point and so has the shortest retention time. The less volatilepentan-1-ol has the
longest retention time.

Elution of Column may be Isothermal (Elution of mobile phase is remain constants temperature
throughout the process) or Gradient (Elution of mobile phase at different temperature normally from low
to higher throughout the process)

Different detector used in GC

Flame Ionization Detector (FID):

Mechanism: compounds are burned in a hydrogen-air flame. Carbon containing compounds produce ions
that are attracted to the collector. The number of ions hitting the collector is measured and a signal is
generated.

Selectivity: compounds with c-h bonds. A poor response for some non-hydrogen containing organics
(e.g., hexachlorobenzene).

Sensitivity: 0.1-10 ng

Linear range: 105-107

Gases: combustion - hydrogen and air; makeup - helium or nitrogen

Temperature: 250-300c,and 400-450c for high temperature analyses.

Nitrogen Phosphorus Detector (NPD):

Mechanism: compounds are burned in a plasma surrounding a rubidium bead supplied with hydrogen and
air. Nitrogen and phosphorous containing compounds produce ions that are attracted to the collector. The
number of ions hitting the collector is measured and a signal is generated.
Selectivity: nitrogen and phosphorous containing compounds

Sensitivity: 1-10 pg

Linear range: 104-10-6

Gases: combustion - hydrogen and air; makeup - helium

Temperature: 250-300c

Electron Capture Detector (ECD):

Mechanism: electrons are supplied from a 63ni foil lining the detector cell. A current is generated in the
cell. Electronegative compounds capture electrons resulting in a reduction in the current. The amount of
current loss is indirectly measured and a signal is generated.

Selectivity: halogens, nitrates and conjugated carbonyls

Sensitivity: 0.1-10 pg (halogenated compounds); 1-100 pg

(nitrates); 0.1-1 ng (carbonyls)

Linear range: 103-104

Gases: nitrogen or argon/methane

Temperature: 300-400c

Thermal conductivity detector (TCD):

Mechanism: a detector cell contains a heated filament with an applied current. As carrier gas containing
solutes passes through the cell, a change in the filament current occurs. The current change is compared
against the current in a reference cell. The difference is measured and a signal is generated.

Selectivity: all compounds except for the carrier gas

Sensitivity: 5-20 ng

Linear range: 105-106

Gases: makeup - same as the carrier gas

Temperature: 150-250c
Flame photometric detector (FPD):

Mechanism: compounds are burned in a hydrogen-air flame. Sulfur and phosphorous containing
compounds produce light emitting species (sulfur at 394 nm and phosphorous at 526 nm). A
monochromatic filter allows only one of the wavelengths to pass. A photomultiplier tube is used to
measure the amount of light and a signal is generated. A different filter is required for each detection
mode.

Selectivity: sulfur or phosphorous containing compounds. Only one at a time.

Sensitivity: 10-100 pg (sulfur); 1-10 pg (phosphorous)

Linear range: non-linear (sulfur); 103-105 (phosphorous)

Gases: combustion - hydrogen and air; makeup - nitrogen

Temperature: 250-300c

Photoionization Detector (PID):

Mechanism: compounds eluting into a cell are bombarded with high energy photons emitted from a lamp.
Compounds with ionization potentials below the photon energy are ionized. The resulting ions are
attracted to an electrode, measured, and a signal is generated.

Selectivity: depends on lamp energy. Usually used for aromatics and olefins (10 ev lamp).

Sensitivity: 25-50 pg (aromatics); 50-200 pg (olefins)

Linear range: 105-106

Gases: makeup - same as the carrier gas

Temperature: 200c

Electrolytic conductivity detector (ELCD):

Mechanism: compounds are mixed with a reaction gas and passed through a high temperature reaction
tube. Specific reaction products are created which mix with a solvent and pass through an electrolytic
conductivity cell. The change in the electrolytic conductivity of the solvent is measured and a signal is
generated. Reaction tube temperature and solvent determine which types of compounds are detected.

Selectivity: halogens, sulfur or nitrogen containing compounds. Only one at a time.

Sensitivity: 5-10 pg (halogens); 10-20 pg (s); 10-20 pg (n)

Linear range: 105-106 (halogens); 104-105 (n); 103.5-104(s)

Gases: hydrogen (halogens and nitrogen); air (sulfur)

Temperature: 800-1000c (halogens), 850-925c (n), 750-825c (s)

Mass Spectrometer (MS):

Mechanism: the detector is maintained under vacuum. Compounds are bombarded with electrons (ei) or
gas molecules (ci). Compounds fragment into characteristic charged ions or fragments. The resulting ions
are focused and accelerated into a mass filter. The mass filter selectively allows all ions of a specific
mass to pass through to the electron multiplier. All of the ions of the specific mass are detected. The mass
filter then allows the next mass to pass through while excluding all others. The mass filter scans stepwise
through the designated range of masses several times per second. The total number of ions are counted
for each scan. The abundance or number of ions per scan is plotted versus time to obtain the
chromatogram (called the tic). A mass spectrum is obtained for each scan which plots the various ion
masses versus their abundance or number.

Selectivity: any compound that produces fragments within the selected mass range. May be an inclusive
range of masses (full scan) or only select ions (sim).

Sensitivity: 1-10 ng (full scan); 1-10 pg (sim)

Linear range: 105-106

Gases: none

Temperature: 250-300c (transfer line), 150-250c (source)

MCQ

Q1. Characteristic feature of any form of chromatography is the ___

A. Use of molecules that are soluble in water.
B. Use of an inert carrier gas.
C. Calculation of an Rf value for the molecules separated.
D. Use of a mobile and a stationary phase.
Q2. A student sets up a paper chromatogram and places a spot of food dye on the origin. After six
minutes the solvent has moved 12 cm and a blue spot has advanced 9 cm. After fourteen minutes the
solvent has advanced a further 8 cm. How many cm from the origin is the blue spot likely to be
A. 26
B. 8
C.18
D.15
Q3. Thin layer chromatography can be used to distinguish between different amino acids. If a particular
amino acid has low solubility in the mobile phase used, then the amino acid
A. Will have a low Rf value.
B. Will spend more time dissolved in the mobile phase than attached to the stationary phase.
C. Will move at a speed close to that of the solvent.
D. Must have a high molecular mass.
Q4.Which of the following is the most suitable gas to use as a carrier gas in a gas chromatogram?
A. Helium
B. Oxygen
C. Methane
D. Carbon dioxide
Q5. A new youth drink contains sugar, salt, alcohol and vitamin C. A gas chromatogram could be used
to determine the
A. Alcohol content only.
B. Alcohol, sugar and vitamin C content only
C. Concentration of all ingredients in the drink.
D. Alcohol and sugar content only.
Q6. Which of the following statements about paper and gas chromatography is correct?
A. The Rf and Rt values of a substance are determined solely by the interaction of the substance with the
stationary phase.
B. A substance with a long retention time in gas chromatography is likely to have a high Rf value in
paper chromatography.
C. A high Rf value is indicative of a substance that adsorbs strongly onto the stationary phase
D. A long retention time in gas chromatography is indicative of a substance with a strong adsorption on
to the stationary phase.
Q7Paper chromatography is
A. Partition Chromatography
B. Adsorption chromatography
C. Electrical mobility of ionic species
D. None of the above.
Q8. In reverse phase chromatography, the stationary phase is made
A. Non-polar
B. Polar
C. Either non-polar or polar
D. Semi polar
Q9. Ion exchange chromatography is based on the
A. Electrostatic attraction
B. Electrical mobility of ionic species
C. Adsorption chromatography
D. Partition chromatography
Q10. Which would be best to separate a protein that binds strongly to its substrate?
A. Gel filtration
B. Affinity chromatography
C. Cation exchange
D. Anion exchange
Q11. The composition of Silica gel G is
A. Silica gel without binder
B. Silica gel + CaSO4
C. Silica gel + alumina
D. silica gel + MgSO4
Q12. Retaintion factor or Retardation factor (Rf) value ranges from
A. 0 to 1
B. 0 to 2.0
C. +2 to -2
D. +1 to -1
Q13. Sucrose can be determined after silylation using which chromatographic technique
A. HPLC
B. Gel chromatography
C. Gas liquid chromatography
D. Paper chromatography
Q14. Q14. Which of the following can not be used as carrier gas in GC?
A. Hydrogen
B. Nitrogen
C. Helium
D. Oxygen
Q15. Retention time is defined as
A. Time needed for an individual analyte to be elute 50%
B. Time needed for all solutes in a sample to be eluted
C. Time needed for complete elution of analyte from the Column
D. Time needed for complete elution of analyte from the Detector
Q16. For better resolution which category of whattman filter paper is used
A. Grade No 20
B. Grade No 4
C. Grade No 540
D. Grade No 3
Q17. Which of the following is used as a detectors in GC
A. TCD & FID
B. AID & ECD
C. Both A & B
D. Katherometer
E. All the Above
Q18. LALLS Detectors used in
A. Gas Chromatography
B. Column Chromatography
C. Size Exclusion Chromatography
D. Ion Exchange Chromatography
Q19. Which type of chromatographic process requires that when materials that cannot be readily
dissolved in a solvent for chromatography need to be heated or pyrolyzed to a high temperature?
A. Gas chromatography
B. High-performance liquid chromatography
C. Thin-layer chromatography
D. Pyrolysis can be used for any and all chromatographic processes
Q20. The higher the solubility of a gas in a liquid, the greater the tendency of the gas molecules to:
A. Remain in the liquid phase.
B. Move from a liquid phase to a gaseous or vapor phase
C. Move from a liquid phase to a solid state
D. Disperse.
Q21. By adding SDS (sodium dodecyl sulfate) during the electrophoresis of proteins, it is possible to:
A. Determine a proteins iso electric point
B. Determine an enzymes specific activity
C. Determine the amino acid composition of the protein.
D. Separate proteins exclusively on the basis of molecular weight
Q22. Nucleic acid can be separated on
A. Silica gel Chromatography
B. SDS-PAGE
C. Agarose Gels
D. Polythelene Gel
E. All the above
Q23. Which detector is preferably used in for the identification of halogenated compounds?
A. Ultraviolet detector
B. Electron Capture detector
C. Thermal conductivity
D. Mass spectrophotometer-detector
Q24. The following is not an example of Partition chromatography
A. TLC
B. Gas Chromatography
C. Paper Chromatography
D. High Performance Liquid Chromatography
E. Both B & D