Chromatography

Lecture 2
 LEARNING Outcomes

After studying this topic, you should be able to :

1. Understand the principle of chromatography
2. Identify the basic components of a chromatography system
3. Understand the purpose of chromatography
4. Describe the different ways to classify chromatographic
techniques
5. Know applications of chromatography
6. Describe different types of chromatography (principle,
phases, method & their application).
7. Calculate the Rf value
Chromatography

• Chromatography (derived from two Greek words "Chroma"

meaning color and "graphein" meaning to write) is the collective
term for a group of laboratory techniques used for the separation
and analysis the components of a mixture.

• This separation is achieved based on the differential distribution

and interaction between the components of the mixture and the
two phases of chromatography

• The first phase is fixed (stationary phase) while the second (the
mobile phase) moves through it in a definite direction.
Basic components of chromatography

• Mobile phase (gas or liquid): the phase which moves

through the stationary phase in a definite direction

• Sample: complex mixture being separated or analysed,

maybe carried by the mobile phase

• Stationary phase (solid or liquid): is the substance that

fixed in place through which the mobile would travel

• Column/support: holding stationary phase

• Eluate: separated components

 Purpose of Chromatography

• Analytical – quantitative and qualitative

analysis

• Preparative - purify and collect one or more

components of a sample in reasonable amounts
Classifications of chromatography

There are different ways for classifying chromatographic

techniques:

1. According to the type of mobile phase

2. According to the packaging/shape type of the stationary

phase

1. According to the mode of separation

1. Based on the type of the mobile phase

• Two forms:
A. Liquid chromatography (LC)
• Here the mobile phase is liquid
• The stationary phase can be either solid (Liquid-solid chromatography)
or liquid (Liquid-Liquid chromatography).

B. Gas chromatography (GC)

• Here the mobile phase is an inert gas (nitrogen or helium)
• The stationary phase is either solid (Gas–Solid Chromatography) or
liquid (Gas-Liquid Chromatography).
2. Based on the packaging/shape type of the
stationary phase

• The chromatography is classified into:

A. Columnar or Column Chromatography
• the stationary phase is packed in a column
(glass/plastic/stainless steel)

B. Planar or Plane Chromatography

• the stationary phase is in the form of a sheet or layer
3. Based on modes of Separation

A. Adsorption chromatography
B. Partition chromatography
C. Steric chromatography
D. Ion-exchange chromatography
E. Affinity chromatography
APPLICATIONS OF CHROMATOGRAPHY
• The chromatographic technique is used for the separation of
amino acids, proteins & carbohydrates.
• It is also used for the analysis of drugs, hormones, vitamins
• Helpful for the qualitative & quantitative analysis of complex
mixtures.
• The technique is also useful for the determination of molecular
weight of proteins.
 Uses for Chromatography
Real-life examples of uses for chromatography:
•Hospital – detect blood or alcohol levels in a patient’s blood sample.
•Pharmaceutical Company – determine amount of each chemical
found in new product
• Law Enforcement – to compare a sample found at a crime scene to
samples from suspects

• Environmental Agency – determine the level of pollutants in the

water supply

• Manufacturing Plant – to purify a chemical needed to make a

product
 Types of Chromatography

• There are following types of Chromatography

➢ Paper Chromatography
➢ Thin Layer Chromatography(TLC)
➢ Ion Exchange Chromatography
➢ Gel Filtration Chromatography
➢ Gas Liquid Chromatography
➢ Affinity Chromatography
➢ High performance liquid chromatography
Type of chromatography Mobile Stationary

Paper chromatography Air, alcohol Filter paper,

cellulose

Thin Layer Chromatography Hexane, ether Silica gel,

petroleum, alumina,
alcohol. polyamide

Gas chromatography He, N2 Squalene,

(GC) apezion,
carbowax M
High Performance Liquid carbon Licosorb,
Chromatography tetrachloride, Silicone
ethanol,
methanol, air
 A. Adsorption chromatography
• Also known as Liquid-Solid
chromatography
• Stationary phase: Solid that attracts
the analyte within the sample
-acidic polar (e.g. silica gel)
-Basic polar (e.g. alumina)
-nonpolar (e.g. charcoal)

• Mobile phase: can be a single

solvent or a mixture of two or more
solvents, depending on the analytes
to be desorbed
• Mechanism of separation: based
on the competition between the
sample and the mobile phase for
adsorptive sites on the solid
stationary phase
A. Adsorption chromatography

• There is an equilibrium of solute molecules being adsorbed to

the solid surface or dissolved in the mobile phase
• The molecules that are most soluble in the mobile phase move
fastest; the least soluble move slowest
• Solubility in the mobile phase is the factor that affects speed of
separation
• Liquid–solid chromatography is not widely used in clinical
laboratories because of technical problems with the preparation
of a stationary phase that has homogenous distribution of
adsorptive sites.
B. Partition chromatography

• Liquid-Liquid chromatography

• The two phases are immiscible

solvents, one is aqueous (polar)
and the other is organic (nonpolar)

• Mechanism of separation:
Separation of solute is based on
relative solubility or partitioning in
the two solvents
B. Partition chromatography
C. Steric Exclusion Chromatography

• A variation of liquid–solid
chromatography
• Stationary phase- porous
material packed in a column
• Mobile phase: Liquid as
water or dilute alcohol
• Mechanism of separation:
separate solute molecules
based on size and shape
C. Steric Exclusion Chromatography

• Small molecules enter the

pores in the packing and are
momentarily trapped
• Large molecules are
excluded from the small
pores and so move quickly
between the particles
• Intermediate-sized
molecules are partially
restricted from entering the
pores and, therefore, move
through the column at an
intermediate rate that is
between those of the large and
small molecules
 D. Ion exchange chromatography
❑ Stationary phase: Solid resin (consisting of
insoluble polymers of benzene, silicates, or
cellulose derivatives) that carries an
immobilized side chain with a charged
functional group.
❑ Mobile phase: Liquid containing electrolytes
❑ Mechanism of separation: Solute ions of
charge opposite to the fixed ions are attracted
to the resin by electrostatic forces & replace
the mobile counterions, so ionic molecules are
separated according to their charge
❑ These are either cation or anion exchangers
based on the charge attached to the resin
Cationic exchanger

- The column carries a sulfonate functional group (-ve)

- Here its made with exchangeable cationic counter ions (H+
ions usually) that are loosely held and free to react.
- Used to remove or separate cations (+ve species out of the
sample solution)
- Once a cation comes in contact with the resin, it replaces the
H+ ions
Anionic exchanger

- The column carries a diethylamine functional group (+ve)

- Here its made with exchangeable anionic counter ions (OH-
ions usually) that are loosely held and free to react.
- Used to remove or separate anions (-ve species out of the
sample solution)
- Once an anion comes in contact with the resin, it replaces the
OH- ions
Applications of Ion Exchange Chromatography
1 Water softening: Removal of Ca2+, Mg 2+ & other multivalent
cations causing hardness of water by filtration through a layer of
strong cationic exchanger.
2 Water deionization: Removal of ions dissolved in water by mixed
cationic/anionic resins. Displaced protons and hydroxyl ions
combine to form water.

3 Separation of electrolytes from non-electrolytes.

4 To separate mixtures of charged molecules, such as amino acids
5 To concentrate dilute ion solution.
 E. Affinity chromatography

• It uses the affinity of analyte to

specific ligandssuch as enzymes.
• Stationary phase: Solid on which
specific ligand is attached

• Mobile phase: Liquid or gas

• Mechanism of action: selective non-
covalent interaction between an analyte
and specific molecules, referred to as
ligands.
• The immobilized ligands act as
molecular fish-hooks & selectively pick
up desired proteins while the remaining
protein pass through the column
APPLICATIONS OF CHROMATOGRAPHY
• The chromatographic techniques are used for the separation of
amino acids, proteins & carbohydrates.
• They are also used for the analysis of drugs, hormones,
vitamins
• Helpful for the qualitative & quantitative analysis of complex
mixtures.
• These techniques are also useful for the determination of sizes
of proteins.
Chromatographic procedures

1. Thin-layer chromatography (TLC)

2. High-Performance Liquid Chromatography (HPLC)
3. Gas chromatography (GC)
Thin-layer chromatography (TLC)

❑ The stationary phase: is a thin

layer of a polar adsorbent
(usually silica gel or alumina)
coated on a glass or plastic
plate.
❑ The mobile phase (eluent): is a
non-polar liquid which travels up
the stationary phase, carrying
the samples with it.
❑ Mechanism of separation:
adsorption mechanism
Thin-layer chromatography (TLC)

❑ Principle of TLC: Components

of the samples will separate on
the stationary phase according
to how much they adsorb on the
stationary phase versus how
much they dissolve in the mobile
phase.
❑ This generate chromatogram:
visual output of the
chromatograph
 General procedure of TLC:
1. A small amount of the mixture to be analyzed is spotted near the bottom of
stationary phase .

2. The TLC plate is then placed in a sealed chamber that contain mobile phase
(eluent), sealing will minimize the evaporation of the solvent (mobile phase)
3. The solvent must be below the line at which samples were spotted, to ensure
proper migration of the samples with the mobile phase
3. Eluent slowly migrates up the TLC plate by capillary action to generate a
chromatogram
4. After solvent reaches predetermined height, the chromatogram is removed from
the developing chamber, dried, and the separated components of the mixture are
visualized.
➢ If the spots can be seen, outline them with a pencil.
➢ If no spots are obvious
✓ Holding the plate under a UV lamp. or
✓ Using substances which reacts with samples an aid in the visualization
Interpretation of TLC results
 Practice

Rf•(A)Rf= (A)= = 0.40

2.0 cm
5.0 cm
Solvent Front

Rf•(B)Rf= (B)=
3.0 cm = 0.60
5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Distance A
migrated = 3.0 cm Rf•(C) 0.8 cm = 0.16
Rf= (C)=
5.0 cm

Distance B
migrated = 2.0 cm
R•f (D) 4.0 cm = 0.80
3.0 cm
Rf= (D)=
5.0 cm
Distance C
migrated = 0.8 cm
3.0 cm
0.8 cm
R•f (URf
1) =(U)= = 0.60
Origen
x x x x x 5.0 cm
A B U C D
0.8 cm
Rf (U2) = = 0.16
5.0 cm
Thin-layer chromatography (TLC)

❑ Uses of TLC: testing the purity of compounds and identification

substances, most used as a semiquantitative screening test

❑ Advantages of TLC: relatively quick and requires small quantities of

material.
High-Performance Liquid Chromatography (HPLC)

• Uses high pressure for fast separations

• Uses controlled temperature, in- line detectors, and gradient elution
techniques
• Pressure and temperature are the factors that affect speed of separation
• Components:
• Pumps: force the mobile phase through the column at a much greater
velocity than that accomplished by gravity flow columns
• Columns:
• the stationary phase is packed into long stainless-steel columns
• columns can be put in an oven and heated to enhance the rate of
partition
• The most common material used for column packing is silica gel
• Column packings size varies (3-20mm), where smaller particles mostly
used for analytic separations and larger ones for preparative
separations.
High-Performance Liquid Chromatography (HPLC)

❑ Components:
❑ Sample injectors: A small syringe can be used to introduce the
sample into the path of the mobile phase that carries it into the
column
❑ Detectors:
• Modern HPLC detectors monitor the eluate as it leaves the
column and, ideally, produce an electronic signal proportional
to the concentration of each separated component
• Spectrophotometers that detect absorbances of visible, UV
light or fluorescence are most commonly used
• Amperometric or electrochemical detectors
• Mass Spectrometer detector
24
 High-Performance Liquid Chromatography (HPLC)

❑ Components:
❑ Recorders:
• Is used to record detector signal versus the time
the mobile phase passed through the instrument,
starting from the time of sample injection
• The graph is called a chromatogram
• The retention time is used to identify compounds
when compared with standard retention times that
run under identical conditions
• Peak area is proportional to concentration of the
compounds that produced the peaks

25
High-PerformanceLiquid Chromatography (HPLC)

• Components:

26
 HPLC vs. LC chromatography
The HPLC has the following characteristic:
1. Columns: Small diameter
2. Column packing with very small particles
3. Peak area is proportional to concentration of the compounds that
produced the peaks
4. Detecting very small amounts
5. High resolution
6. Rapid analysis
7. Speed, efficiency, sensitivity and ease of operation
High-Performance Liquid Chromatography (HPLC)

• Uses of HPLC:
1. Analyze drugs and medicines for their purity
2. Vitamins in blood
3. Drug analysis in urine
4. Nutrients in food
 Using HPLC for qualitative measurements

B
A C

A=

B=

C=
Using HPLC for quantitative measurements
 Gas Chromatography (GC)
❑ Used to separate mixtures of compounds that are volatile or can be made volatile
❑ Stationary phase: solid or a thin film of nonvolatile liquid supported on an inert
support

❑ Mobile phase: inert carrier gas (nitrogen, helium or argon)

❑ In gas chromatography (GC), the sample is vaporized and injected onto the head
of a chromatographic column which is packed with the stationary phase
❑ Compounds with higher boiling points will move slowly through the column

❑ Boiling point is the factor that affects speed of separation

 Gas Chromatography (GC)
❑ Elution is done by the flow of an inert gaseous mobile phase.
❑ The mobile phase does not interact with molecule of the analyte; its only
function is to transport the analyte through the column
❑ The effluent passes through a detector that produces an electric signal
proportional to the concentration of the volatile components
❑ As in HPLC, the chromatogram is used both to identify the compounds by
the retention time and to determine their concentration by the area under
the peak
❑ Columns: GLC columns are generally made of glass or stainless steel

❑ Detectors: thermal conductivity (TC) and flame ionization detectors

❑ GLC is commonly used in clinical laboratories

Summary
References:

• Chapters 13-15 of the Laboratory Instrumentation book

• Chapter 6 of Clinical chemistry book by Bishops
• TLC demonstration
• Gas Chromatography
• HPLC