CHROMATOGRAPHY

Module 4
 Chromatography
 It originated from the Greek words
“chroma” meaning “color” and
“graphein” meaning “to write”
 It was coined by the Russian
botanist “Mikhail Semyonovich
Tsvet [Tswett]” during his research
about chlorophyll where he
separated the different plant
pigments
 Chromatography
Definition
 Science of the study of
separation of molecules based
on differences in their structure
and/or composition
 It is a collective term for a family
of laboratory techniques for the
separation of mixtures
 Chromatography
Principle
 Itinvolves passing a mixture
dissolved in a “mobile phase”
through a “stationary phase” which
separates the analyte to be
measured from the other molecules
in the mixture
CHROMATOGRAPHY: THEORY

 MOBILE PHASE
A GAS OR LIQUID THAT PASSES
THROUGH THE COLUMN.

 STATIONARY PHASE
A SOLID OR LIQUID THAT DOES NOT
MOVE. IT REFERS TO THE
CHROMATOGRAPHIC SUPPORT.
THE CHROMATOGRAPHIC SYSTEM

 T

STATIONARY
PHASE
(SILICA PLATE)

MOBILE PHASE
(LIQUID)
Preparative vs Analytical
Chromatography
◦ Preparative chromatography
 It seeks to separate the components of
a mixture for further use and thus is a
form of purification
◦ Analytical chromatography
 It normally operates with smaller
amounts of materials and seeks to
measure the relative proportions of
analytes in mixtures
PRINCIPAL OBJECTIVES
OF CHROMATOGRAPHY
 Principal Objectives
 Resolution of mixtures into
constituent parts
 Determination of homogeneity
 Comparison of substances
suspected of being identical
 Purification
 Principal Objectives
 Concentration of substances from
dilute solutions
 Identification and control of
technical products
 Quantitative separation from
complex mixtures
 Indication of molecular structure
CHROMATOGRAPHY
THEORIES
CHROMATOGRAHY: THEORIES

 RATE THEORY

 PLATE THEORY

BOTH EXHIBITS THE CAPILLARY

ACTION, TO MOVE THE SOLVENT
THROUGH THE STATIONARY PHASE.
RATE THEORY AND PLATE THEORY
HOW IT WORKS?

 THE MOBILE PHASE MOVES THROUGH THE

STATIONARY PHASE, PICKING UP THE
COMPOUNDS TO BE TESTED.

 AS THE MOBILE PHASE TRAVELS THROUGH

THE STATIONARY PHASE, IT TAKES THE
COMPOUNDS WITH IT.
 RATE THEORY AND PLATE THEORY
HOW IT WORKS?

 AT DIFFERENT POINTS IN THE STATIONARY

PHASE, THE DIFFERENT COMPONENTS OF
THE COMPOUND ARE GOING TO BE
ADSORBED; AND ARE GOING TO STOP (AT A
SPECIFIC DISTANCE) MOVING THRU THE
MOBILE PHASE.
 RETENTION: DEFINITION

 MEASURE OF SPEED AT WHICH A SUBSTANCE

MOVES IN A CHROMATOGRAPHIC SYSTEM.

 IN CONTINUOUS DEVELOPMENT SYSTEMS (HPLC & GC),

WHERE THE COMPOUNDS ARE ELUTED WITH AN ELUENT, IT
IS EXPRESSED AS RETENTION TIME (Rt).

 IN INTERRUPTED DEVELOPMENT SYSTEMS ( TLC & PC ), IT

IS EXPRESSED AS RETENTION FACTOR (Rf).
PRINCIPLES
OF
SEPARATION
 Principles of Separation
 Adsorption
◦ Retention and separation
depends on the ability of the
atoms on the surface to remove
analytes from the mobile phase
and adsorb them temporarily by
means of electrostatic forces
 Principles of Separation
 Partition
◦ Retention and separation occur
due to the relative solubility of
the analytes in the two fluids as
determined by their partition
coefficients
 Principles of Separation
 Ion-exchange
◦ Retention and separation is
mainly due to the electrostatic
bonds with the functional groups
 Principles of Separation
 Molecular exclusion
›Also known as “size exclusion”,
“gel permeation” or “gel
filtration”
›Retention and separation
depends on the differential
migration of solute molecules
based on molecular size
DIFFERENT
CHROMATOGRAPHIC
TECHNIQUES
 Based on Chromatographic Bed
Shape
◦ Column Chromatography
◦ Planar Chromatography
 Paper chromatography
 Thin Layer chromatography
 Based on Physical State of Mobile
Phase
◦ Gas chromatography
◦ Liquid chromatography
 HPLC
APPLICATIONS
OF
CHROMATOGRAPHY
CHROMATOGRAPHY: APPLICATIONS

 LIQUID CHROMATOGRAPHY
TEST WATER SAMPLES FOR
POLLUTION.
CHROMATOGRAPHY: APPLICATIONS

 GAS CHROMATOGRAPHY
1. BOMB DETECTION IN AIRPORTS
CHROMATOGRAPHY: APPLICATIONS

GAS CHROMATOGRAPHY
2. IDENTIFY / QUANTIFY DRUGS &
ALCOHOL.
CHROMATOGRAPHY: APPLICATIONS

3. IN FORENSICS TO COMPARE
FIBERS FOUND ON A VICTIM.
CHROMATOGRAPHY: APPLICATIONS

 THIN LAYER CHROMATOGRAPHY

1. DETECTION OF PESTICIDES &
INSECTICIDES IN FOOD.
2. IN FORENSICS TO ANALYZE DYE
COMPOSITION OF FIBERS.
CHROMATOGRAPHY: APPLICATIONS

 PAPER CHROMATOGRAPHY
1. SEPARATION OF AMINO ACIDS AND
ANIONS.

2. RNA FINGERPRINTING
CHROMATOGRAPHY: APPLICATIONS

PAPER CHROMATOGRAPHY
3. SEPARATION AND TESTING OF
HISTAMINES & ANTIBIOTICS.
COLUMN
CHROMATOGRAPHY
 Column Chromatography
 Is a separation technique in
which the stationary bed is within
a tube
 It is a method used to purify
individual chemical compounds
from mixtures of compounds
CC Components
 Chromatographic Column
◦ The simplest type is consists of a
suction flask and a cylindrical glass
or quartz constricted at one end with
a stopcock or flow regulator.
◦ The size of the column is determined
by the quantity and adsorbability of
the substance being separated.
CC Components
 Stationary Phase
› Adsorbent or packing materials
placed inside the column to adsorb
the sample
› Usually a solid material, finely
ground powders or gels which are
microporous for an increased
surface
CC Components
 Stationary Phase
› Common Adsorbents
 Silica gel
 Alumina
 Calcium carbonate
 Purified siliceous earth
 Cellulose powder
 Diatomaceous earth (celite)
 Cross-linked dextrans (SephadexLH
20)
 CC Components
 Mobile Phase
◦ Is either a pure solvent or a
mixture of different solvents
◦ Alcohols and Amides – nonpolar
materials
◦ Purified water – polar materials
 Sample Preparation
and Application
› The adsorbent is introduced into the
column either as a dry powder or as a
slurry suspended in the mobile phase
›Benzene (hydrophobic solvent)
›Alcohol (hydrophilic solvent)

› In either case, care must be taken to

avoid air bubbles
 Evaluation of Chromatogram
› For colored samples that fluoresce
under UV light, the adsorbent column is
forced intact from the tube and the
fractions are easily divided by a knife.
› For colorless samples, visualization is
done by first spraying or painting the
extrude column with a color forming
agent.
› For radioactive samples, their position
in the column is determined using a
Geiger-muller counter.
Flash Column
Chromatography
◦ A modified version of column
chromatography introduced by W.C.
Still in 1978
◦ It is very similar to the traditional
column chromatography, except for
that the solvent is driven through the
column by applying positive
pressure
PLANAR
CHROMATOGRAPHY
 Planar Chromatography
 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
◦ A layer of solid particles spread on
support such as a glass plate
PAPER
CHROMATOGRAPHY
 Paper Chromatography
 A method invented by the British
biochemists, Archer John Porter
Martin and Richard Laurence
Millington Synge
 An analytical technique for
separating and identifying
mixtures that or can be colored,
especially pigments
 PC Components
 Stationary Phase
◦ It is consists of a sheet of paper with
controlled texture and thickness
usually made up of cellulose [filter
paper].
 PC Components
 Mobile Phase
◦ The mobile phase used in paper
chromatography may include any of
the following:
 Mixture of alcohol and water with added
ammonia or acetic acid
 Chloroform
 Benzene
 Cyclohexane
 Sample Preparation
and Application
› Samples are applied as a solution in volatile
solvent [usually in quantities of 0.1 to 1000
mcg]
› Samples are applied to a strip of
chromatography paper at origins using
capillary pipettes or microliter syringe
 For ascending chromatogram, the spots are
applied at 3-5 cm from the lower edge.
 For descending chromatogram, the spots are
applied at 7-9 cm from the upper edge.
 For radial chromatogram, the spots are applied on
a circle with a radius of 1-3 cm.
 Evaluation of
Chromatography
◦ Computation of the Rf values and
comparison with standards
 Rf value =Distance traveled solute
Distance traveled solvent

◦ Elution – cutting the spot and

soaking the paper in an appropriate
solvent
THIN LAYER
CHROMATOGRAPHY
Thin Layer Chromatography
 A widely employed technique similar
to paper chromatography
 It involves spotting of sample or a
mixture of samples at one end of an
adsorbent-coated glass plate followed
by passage of a solvent through the
adsorbent for the purpose of
separating the components of a
mixture
Advantages of TLC
◦ Rapid
◦ Sensitive
◦ Excellent resolution
◦ Simplicity
◦ Cheap
 TLC Components
 Stationary Phase
› It consists of a thin layer of
adsorbent material immobilized
onto flat, inert carrier sheet.
› TLC Plates
 TLC Components
› TLC Plates
 TLC plates are made by mixing the
adsorbent, such as silica gel with a small
amount of inert binder like calcium sulfate
[gypsum] and water.
 The mixture is spread as thick slurry on an
unreactive carrier sheet, usually glass, thick
aluminum foil or plastic, and the resultant
plate is then dried and activated by heating
in an oven for thirty minutes at 110C.
 TLC Components
 Mobile Phase
› The common solvents used are the following:
Heptane Ethyl ether Acetonitrile
Hexane Chloroform Pyridine
Isoctane Acetone Ethylene chloride
Cyclohexane Ethanol
Methanol CCl4 1-Propanol
Acetic acid Toluene Dioxane
Water Benzene Ethyl acetate
 TLC Components
 Mobile Phase
› For mixtures of unknown composition,
benzene or chloroform with 10% ethanol
is the best solvent for explorotatory runs
Evaluation of Chromatogram
◦ Once the chromatogram has been
developed, the spots must be made
visible in order to determine the
Rf values.
Special Detection Methods
 Charring
Involves the spraying of
concentrated sulfuric acid and
heating the plate.
The result is seen by the charring
of the spots.
Special Detection Methods
 Use of Iodine Vapor
In this method, the chromatogram
is placed in a closed container
holding a few iodine crystals.
The sample spots react with the
iodine vapor and form brown
spots.
The reaction is reversible.
Special Detection Methods
 Examination Under UV
Radiation
Useful for compounds that
fluoresce.
Two UV light sources are useful
and commercially available, these
are the UV short-wave [254 nm]
and long-wave [360nm].
 SPECIFIC SPECIFIC
VISUALIZING AGENTS COMPOUNDS IDENTIFIED

NINHYDRIN AMINO ACIDS

RHODAMINE B LIPIDS

ANTIMONY CHLORIDE STEROIDS

TERPINOIDS
SULFURIC ACID + HEATING UNIVERSAL VISUALIZING
AGENT FOR MOST ORGANIC
SUBSTANCES

POTASSIUM PERMANGANATE HYDROCARBONS

IN SULFURIC ACID
ANISALDEHYDE IN SULFURIC CARBOHYDRATES
ACID
BROMINE VAPOR OLEFINS
 Derivatization
Functional Groups Reagents Color Produced

Acids Bromcresol green Yellow

Aldehyes and Ketones 2,4- Yellow

dinitrophenylhydrazine

Amines and amino Ninhydrin Fluorescent

acids
Alkaloids Mercuric nitrate Yellow to brown

Barbiturates Diphenylcarbazone Purple

Carbohydrates Aniline phthalate Gray-black

Lipids Bromthymol blue Light green

Steroids Antimony trichloride Various

THIN LAYER CHROMATOGRAPHY:
DRUGS ANALYZED:

 ANALGESICS
 ANTIPYRETICS
ASPIRIN, PHENACETIN, ACETAMINOPHEN

 ANTI-INFLAMMATORY DRUGS
 URICOSURIC DRUGS
 CAFFEINE AND CAFFEINE-CONTAINING DRUGS
GAS
CHROMATOGRAPHY
 GAS Chromatography
 Also known as “gas-liquid
chromatography” or “gas-liquid
partition chromatography”
 A separation technique in which
the mobile phase is a gas
 It is used in the analysis of
gaseous and volatile substances
 GC ADVANTAGES

 EXCELLENT SEPARATION, USUALLY LESS THAN 60

SECONDS.

 SENSITIVITY & SPEED ARE EXTRAORDINARY, WITH 10

TO 12 GRAM SAMPLE BEING DETECTABLE FOR MANY
SUBSTANCES.
 GC ADVANTAGES

 GC CAN BE RUN 1,000 TIMES FASTER THAN LC.

 LARGER PREPARATIVE GLC’S CAN BE USED FOR

PURIFICATION OF SAMPLES.
 GC Components
 Mobile Phase
›This is the carrier gas
◦ Carrier Gases Employed in GC
 Helium
 Nitrogen
 Hydrogen
 Argon
 Air
GC Components
› Considerations in the Choice of
Carrier Gas
 Safety
 Purity
 Inertness
 Availability
 Cost
 Detector to be used
 Sample’s matrix
 GC Components
 Stationary Phase
◦ It consists of a microscopic layer of
liquid or polymer of an inert solid
support inside glass or metal tubing
[a column]
◦ It functions in separating the different
components of the sample
 GC Components
 GC Column
◦ The “heart” of gas chromatography
◦ These are long tubes packed with a
sorbent material
 GC Components
 GC Column Types
 Packed columns
 Capillary columns
GC System
 GC System:
Components and Functions
› Carrier Gas Tank
 A chamber made of stainless steel that
supplies the carrier gas needed in the
analysis

› Pressure Regulator
 A suitable two-stage diaphragm
controlled pressure regulator that
reduces the pressure level compatible
with the requirement of the instrument
 GC System:
Components and Functions
› Flow Controller
 Contained within a thermostated chamber
capable of maintaining a constant
temperature as high as 400C
 Controls gas flow rate, the gas flow rate is
selected to compromise between the length
of the analysis and the level of separation
 The rate at which a sample passes through the
column is directly proportional to the gas flow rate
in the column.
 The higher the flow rates the faster the analysis,
but the lower the separation between analytes.
 GC System:
Components and Functions
› Injector Port
 A small chamber where the sample is
introduce into the system
 The primary requirement of the injection
system is that the sample be vaporized
instantaneously so that a narrow band of
vapor is introduced into the beginning of the
column
 It is equipped with a self-sealing septum
made of rubber or silicone to prevent the
sample from escaping
 GC System:
Components and Functions
›Injector Port
 The choice of the inlet type and injection
technique depends on the type of sample
and solvent matrix.
 Dissolved samples are introduced directly onto
the column via COC injector.
 If a solvent matrix has to be vaporized and
partially removed, a S/SL injector is used.
 Gaseous samples are usually injected using a
gas switching valve system.
 GC System:
Components and Functions
› Column or Column Oven
 The “heart” of the GC system, it is
contained in an oven, the temperature
of which is precisely controlled.
 Its sole function is to maintain the
constancy and uniformity of the column
temperature at the desired value.
 An airbath is used to maintain this
requirement.
 GC System:
Components and Functions
› Column or Column Oven
 Column temperature is selected to
compromise between the length of the
analysis and the level of separation
 The rate at which a sample passes through the
column is directly proportional to the temperature
of the column.
 The higher the column temperature, the faster
the sample moves through the column.
 However, the faster a sample moves through the
column, the less it interacts with the stationary
phase and the less analytes are separated.
GC Column Oven
 GC System:
Components and Functions
› Detector
 A number of detectors are used in GC.
 The most common detectors are the
following:
 Thermal Conductivity Detector [TCD]
 A universal detector
 Simple, inexpensive and nondestructible to the
sample
 Flame Ionization Detector [FID]
 Highly sensitive detector
 Almost a universal detector
 GC System:
Components and Functions
› Other Detectors
 Discharge ionization detector [DID]
 Electron capture detector [ECD]
 Flame photometric detector [FPD]
 Hall electrolytic conductivity detector [EICD]
 Nitrogen phosphorus detector [NPD]
 Mass selective detector [MSD]
 Photo ionization detector [PID]
 Pulsed discharge ionization detector [PDD]
 Thermal energy analyzer [TEA]
 GC System:
Components and Functions
› Recorder/Integrator
 Used to graphically reproduce the
output of the detector and record the
resulting “chromatogram”
 The position of the peak on the
chromatogram is measured in terms of
retention
 GC System:
Components and Functions
 Retention time [Rt]
 The time required by an average molecule
of component to pass from the injector
port through the column to the detector
 Retention volume [Rv]
 The volume of the carrier gas necessary to
carry an average molecule of the
component from the point of injection to
the detector
 GC Applications
 In analytical chemistry and biochemistry
 In petrochemical environmental
monitoring
 In chemistry research
 Measurement of toxic substances in air,
water and soil
 In drug quality control, to assure the
quantitative/qualitative features of
pharmaceuticals
 Assay of volatile oil content
LIQUID
CHROMATOGRAPHY
 LIQUID Chromatography
 Liquid chromatography 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 LC that generally utilizes
very small packing particles and a
relatively high pressure is referred to
as high performance liquid
chromatography [HPLC]
HIGH PRESSURE LIQUID
CHROMATOGRAPHY
 High Performance Liquid
Chromatography
 Also known as “high pressure liquid
chromatography”
 It is a form of column chromatography
used frequently in biochemistry and
analytical chemistry to separate,
identify and quantify compounds.
HPLC Advantages
◦ Greater speed
◦ Precision
◦ Accuracy
◦ Ease of separation
HPLC Prerequisite
◦ Sample must be soluble with
solvent (must be HPLC grade)
HPLC Applications
◦ Nonvolatile substances
◦ Substances with high polarity or
highly tonic
◦ Substances with high molecular
weight
◦ Thermally unstable and
decomposable substances
 HPLC Components
 Mobile Phase
› Common solvents used include any miscible
combinations of water or various organic
liquids [methanol and acetonitrile]
› Water may contain buffers or salts to assist in
the separation of the analyte components or
compounds such as trifluoroacetic acid (TFA)
which acts as an ion pairing agent
› One important requirement is that the solvents
used must be degassed first
 HPLC Components
 Stationary Phase
› It is contained in the column and it
consists of packing materials that are of
two types:
 Porous Packing Materials
 Superficially Porous Packing Materials
 HPLC System:
Components and Functions
◦ Solvent Reservoir
 A glass or stainless steel container
capable of holding up to a liter of
mobile phase
 HPLC System:
Components and Functions

›Pumps
 A device needed to move the
mobile phase through the column
 Types
 Mechanical pump
 Pneumatic pump
 HPLC System:
Components and Functions
◦ Injection Valve
 A chamber where the solute is
introduced through a self-sealing
rubber or Teflon disc using a
microliter syringe
 HPLC System:
Components and Functions
◦ HPLC Column
 Precolumn or Guard Column
 A solid support coated with a high
percentage of liquid phase than the
analytical column
 Analytical Column
 A stainless steel tube where the actual
separation takes place
 HPLC System:
Components and Functions
›Detector
 A device that measures the
concentration of the sample
injected on the column
 HPLC System:
Components and Functions

 Common Detectors
 UV detectors [for UV emitting compounds]
 Refractive index detector [a universal detector]
 Fluorescence detector [for fluorescent compounds]
 Conductivity detector [for ionic compounds]
 Electrochemical detector [for oxidizable and
reducible compounds]
 Reaction detector [for reactive compounds]
 Radioactivity detector [for radioactive compounds]

 HPLC System:
Components and Functions
◦ Integrators
 Used to graphically reproduce the
output of the detector and record
the result [chromatogram]
HPLC Types
◦ Normal Phase HPLC
 A method that separates analytes based
on polarity and is useful for polar
analytes

◦ Reversed Phase HPLC

 It is used for testing and qualifying drugs
before their release in the market
 HPLC Types
› Size Exclusion HPLC
 It is generally a low resolution chromatography
and is often reserved for the final polishing step
of purification
 It is also useful for determining the tertiary
structure and quaternary structure of purified
proteins
 It is widely used for the molecular weight
determination of polysaccharides
 Based on the European Pharmacopeia, it is the
suggested official technique for the molecular
weight comparison of commercially available
low-MW heparins
HPLC Types
◦ Ion-Exchange HPLC
 Used in purifying water, pre-
concentration of trace components,
ligand-exchange chromatography, ion-
exchange chromatography of proteins
and high pH-anion exchange of
carbohydrates and oligosaccharides
The end!!!