Chromatography

Chapter 8
 Chromatography
• Chromatography is a powerful separation technique that can be applied
in all branches of science.
• Chromatography encompasses a diverse and ranges of methods that
allows separation, identification, and quantification of components, and
can be used;
 To isolate a single bio-molecule from a complex mixture in pure
form.
 To separate complex mixtures (e.g. bio-molecules) into individual
components for analytical or quantitative purpose.
 To eliminate contaminants.
 Principle of Chromatography…

• The principle is based on the distribution of species between a stationary

phase and a mobile (moving) phase.

– Stationary phase:

• The adsorbent is a solid fine particles. The most common stationary

phase for chromatography is silica followed by alumina.

– Mobile phase:

• Sample species mover and could be a liquid or gas.

Types of separation
Separation by partition:
Separation by adsorption: Separating based on two immiscible phases.
Separation based on the solute In liquid-liquid chromatography (LLC), the stationary

being attracted by forces generated phase is coated with a polar substance. (silica or
alumina coated with a monolayer of water or some
at the surface of a solid stationary
other liquid.
phase (the silica material being
Separation by partition is based on relative affinity
modified).
of solute for each phase.
General Scheme of chromatography
 Types of Chromatography
Classification of
chromatography

Planar Column
chromatography chromatography

- Planar Chromatography: silica particles coated/casted on flat surface or the

interstice s of paper. The mobile phase in this technique moves through the

stationary phase by capillary action or under the influence of gravity.

- Column chromatography: the stationary phase is held/packed in a narrow tube

through which the mobile phase is passed through under the use of pressure.
 Types of Chromatography

Planar chromatography

Paper
chromatography

Thin layer
chromatography (TLC)
Planar Chromatography
 Planar chromatography
Paper chromatography and thin layer chromatography (TLC) are analytical
chemistry techniques for separating and identifying mixtures that are or can be
coloured, especially pigments.
Compounds in the sample mixture move at different
rates due to differences in solubility in the solvent
(mobile phase) also due to their attractions to the
paper or the layer (stationary phase).

Solvent
Apply a sample If separation is not occurred
Solvent front
using a capillary completely, employ 2-dimensional
tube/needle, and separation technique; rotate the
allow it to dry paper 900 and repeat the
It is important that the chamber has to be procedure. Use combinations of
air tight to avoid solvent evaporation. different concentrations of solvents
The use of filter paper in the chamber ( e.g. methanol:hexane 1:9 or 2:8)
enhances (saturates) solvent vaporization.
Identification Techniques

 Locations of spots that are not colored in the samples my not be visible
on the TLC plate.
Methods of detection
- Fluorescence, using a UV lamp

- Treat the plates with reagents to develop colors, e.g. use Iodine (I2).

- Other special reagents, such as:

- Rhodamine B used for the visualization of lipids
- Ninhydrine for amino acid detection.
 Thin layer chromatography (TLC)
 It is similar to paper chromatography

 Uses acidic (silica) or basic (alumina) adsorbent layers as a stationary phase

 layer of adsorbent is spread over an inert flat support (glass, aluminum or plastic)
and dried
 Adsorption occurs via H-bonding; hence binding strength depends on polarity of
solute
 Separation of the solute from the adsorbent requires a solvent more polar than
the solute
Advantages over paper chromatography
• Separate different classes of bio-molecules
• Faster flow rates, improved resolution
• Withstand harsh treatments (e.g. sulfuric acid)
• Samples can be recovered from silica
The movement of a compound relative to the movement of the solvent can be
determined from the flow rate (RF).

RF = Distance compound has moved from origin

Distance solvent has moved from origin

Comparison of RF values of sample and standard can be used for identification of species
in the sample.
Column Chromatography
Column chromatography
Mobile phase
Sample

B
Stationary phase/ A
packed column
B
A
B

A B

Separation by a column chromatography involves elution, which is

washing a sample through a column by continuous addition of fresh
mobile phase.
 Column chromatography…
• A column chromatography run is achieved by gravity flow. However the flow
rate can be increased.
– By increasing the flow of the fresh eluent (mobile phase).
– by using a pump (compressed air such as nitrogen and argon). Hence apply
pressure.
• The average rate of time a solute migrates down the column depends on The
fraction of time it spends in the mobile phase.
• Fast moving solutes have a noticeably smaller time frame since they move with
the mobile phase and less strongly retained by the stationary phase.
 Column chromatography
In an automated chromatography, the instrument is consist of a number of
parts:

Mobile phase
Pump Injector
reservoir

Detector Column

Waste
Integrator/printer
 Factors affecting the retention time

Different compounds have different retention times. For a particular compound,

the retention time will vary or may not be reproducible, if :
– The solvent composition

– The pressure used, which affects the flow rate of the solvent

– The nature of the stationary phase (not only what material it is made of, but
also particle size)
– The column temperature is altered.

Hence the conditions used when operating chromatography have to be carefully

controlled if you are using retention times as a way of identifying compounds.
Gas Chromatography
GC instrument
 Gas Chromatography (GC)

• Applicable when components of a vaporized samples is to be separated

• Gaseous mobile phase and a liquid or a solid stationary phase

• The separation mechanism is based on partitioning or adsorption

• Vaporized sample is introduced into the gaseous phase; components then

partition between mobile and stationary phases.
• Volatile components elute first, while components with high affinity to the
stationary phase retained in the column.
• Components move through a GC column as gases, either because they are
normally gases or they can be heated and vaporized into gaseous state.
Types of gas chromatography
Gas-solid chromatography (GSC)

• The mobile phase is gas and stationary phase is solid

• Has limited application due to its semi-permanent retention of active (polar)

molecules and also sever tailing of elution peaks.

– (tailing is a result of non-linear nature of the adsorption process).

• uses for the separation of low molecular mass gaseous species.

Gas-liquid chromatography (GLC)

• The mobile phase is gas and stationary phase is liquid

• Separation by GLC is achieved by partition process where a gaseous carrier and

liquid phase immobilized on the surface of an inner solid packing or on the wall of a
capillary tubing stationary phase is used.
Components of GC
Mobile phase

• The mobile phase (carrier gas) is a gas.

– It must be chemically inert (helium, argon). Helium is the most common

carrier gas.

– The gases are available in pressurized tank.

– Pressure regulators, valves, gauges and flow meters are required to

monitor the flow rate of the gas.

– Often the carrier gas is fitted with a molecular sieve to remove impurities

and water.

– The carrier gas flow can be quantified either by linear velocity expressed in

cm/sec or by volumetric flow rate in mL/min.

Sample injection system
• To achieve high column efficiency, the sample must be of a suitable size (to
avoid overloading) introduced as a ‘plug’.
• The sample port is ordinarily about 50 ⁰C above the boiling point of the least
volatile components of the sample.
• Sample splitter is often needed to deliver a known fraction (1:50 to 1:500) of
the injected sample with the remainder going to waste
Column
Two types of columns are used in GC:

- Packed column
- Length: 1-5 m

- open tabular or capillary column

- Length: up to 100 cm

- Most columns are fabricated from fused silica and stainless steel, although glass and teflon are

used.

- Column temperature is an important factor and must be controlled to precise work (avoid

temperature fluctuation).

- Optimal column temperature depends on the boiling point of the sample and the degree of

separation..
Column

• Optimal resolution is achieved with minimal temperature.

 Temperature equal to or slightly above the average boiling point of

the sample results in a reasonable elution time (2 - 30 min).

if temperature is too high, low boiling components swept

readily through the column too fast without being equilibrated

with the stationary phase.

if the temperature is too low, condensation of some organic

compounds in the liquid stationary phase occurs, resulting

longer retention time

Detection systems

• Ideal GC detectors need to have the following characteristics:

- Adequate sensitivity (especially for small amount samples)
- Good stability and reproducibility
- A linear response to solutes that extends over several orders of
magnitude
- A wide ranges of temperature to at least 400 ⁰C
- A short analysis time independent of flow rate
- Highly reliable, durable and easy to operate
- Non-destructive
GC detectors…
• Flame ionization detector (FID)
• Nitrogen phosphorus detector (NPD)
• Electron capture detector (ECD)
• Thermal conductivity detector (TCD)
• Flame photometric detector (FPD)
• Photoionization detector (PID)
• Electrolytic conductivity detector (ELCD)
Liquid Chromatography
Liquid chromatography

Types of liquid chromatography

– High performance liquid Chromatography (HPLC)
– Ion chromatography
– Size-exclusion chromatography/gel chromatography
– Affinity chromatography
High performance liquid Chromatography (HPLC)

• Most widely used of all off the analytical separation techniques due to:
– Sensitivity
– Readily availability to accurate quantitative determination
– Easy of automation
– Suitability to separate/analyse non-volatile or thermally fragile

components (proteins and drugs)

• The term “high performance” signifies the use of very small particles
packed into a reasonably narrow tube (2-4.6mm i.d.) of short length. As
a result it necessitates the use of higher pressure in order to establish a
flow of mobile phase.
HPLC instrument
HPLC instrument
 Components of HPLC
• Mobile phase reservoirs
o Modern LC’s are equipped with one or more glass reservoirs.
o Inlet filters

 To remove particles and dusts from the solvent.

o Back pressure regulator

 To prevent solvent bubble formation (air bubble creates

noise) until the solvent is completely through the detector.
o Gas sparging

 To force dissolved gasses out of the solvent being stored in

the solvent reservoir.
 Components of HPLC

 Pumping system
o The purpose of the pump is to provide a constant, reproducible
flow of solvent through the column. It should controlled,
reproducible flow delivery of about 1 mL/min for analytical
applications and up to 10 mL/min for preparative application.
o It should yield pulse-free solvent flow, however a HPLC is equipped
with a pulse dumper.
o as the result of the need to use high pressure, HPLC is more
expensive than other types of chromatography systems.
 Components of HPLC
Injectors
– Sample injection is achieved via a syringe (the rheodyne injector is
the standard injector used in HPLC).
– The injector has a load and inject position.
– maximum injection is determined by the loop volume.
– only liquid samples can be injected into the HPLC mobile phase.
 Components of HPLC

The columns
– Constructed from smooth bore stainless steel or heavy walled glass

Analytical columns
– Range from 5-25 cm long, 3-5 mm i.d. and 3-5 µm packing particle
sizes.
– length of column can be increased by connecting columns.
– The most common columns are 10-15 cm, 4.6 mm i.d. and 5 µm
particles.
– Micro-columns 3-7.5 cm, 1-4.6 mm i.d. and 5 µm particles.
advantageous for speed and minimal solvent use.
HPLC columns
Components of HPLC

Guard columns
– It is similar to analytical column, but much shorter and bigger
particle size, hence minimizes back pressure.
– Used to increase the life of the analytical column by not only
removing any particulates and contaminates from the solvent, but
components that bind irreversibly to the stationary phase.
Column temperature control
– Column can be operated at room temperature but needs to be
controlled constantly to obtain reproducible results
 Components of HPLC
 Detectors
- UV-visible: Good quality solvents free of trace absorbing impurities
(analytical grade) are needed to be able to use UV down to 205 nm
or less. UV-visible detector has many useful characteristics, such as
high sensitivity, small sample volumes, non-distractive to samples,
suitable for gradient elution.
- Florescence: very sensitive for florescence active compounds

- Refractive index: not so sensitive, but is a useful universal detector.

all compounds have a property of refracting light in solution.

Other detectors, evaporative light scattering detectors,

electrochemical detection and mass spectrometry.
 Application of HPLC

• It is ideal for the separation and identification of amino acids,

proteins, lipids, carbohydrates, proteins, nucleic acids, steroids,
pigments and pharmaceutical products
• Advantage of HPLC

– Speed (minutes)

– High resolution

– Sensitivity (ng to fg)

– Reproducibility of +/- 1% (not so for LC)

– Accuracy

– Automation
limitation of HPLC
– Cost
– Complexity
– Low sensitivity for some compounds
– Irreversibly adsorbed compounds not detected
– Co-elution difficult to detect
Comparison of HPLC to traditional or classical LC

– High resolution and speed

– Reusability of columns without repacking and regeneration
– Reproducibility can be controlled since the parameters
controlling the efficiency of separation can be controlled
– Instrumentation and data manipulation are fully automated
– It is adaptable to scale-up or preparative systems
End of chapter 8