BASIC

CHROMATOGRAPHY

Tri Rini Nuringtyas

Laboratorium Biokimia
Fakultas Biologi
Separation of Compounds of
Biological Origin

Using:
● Extremes of temperature
● Extremes of pH
● Organic solvents – for protein
● Oxidizing / reducing agents
have to be avoided

Extreme physical conditions may:

● Irreversibly change the structure of the molecules
● Destroy any biological activity
Chromatography
This technique utilizes differences in the basic
physical properties:
● Mass
● Size
● Shape
● Charge
● Adsorption effect
 Definition
a separation technique based on the different
interactions of compounds with two phases,
a mobile phase and a stationary phase, as the
compounds travel through a supporting
medium.
 components
• mobile phase: a solvent that flows through the
supporting medium

• stationary phase: a layer or coating on the

supporting medium that interacts with the
analytes

• supporting medium: a solid surface on which

the stationary phase is bound or coated
Kinds of Chromatography
● Gel Filtration
● Adsorption Chromatography
● Ion Exchange Chromatography
● Partition Chromatography
● Paper Chromatography
● Thin-layer Chromatography
● Gas Chromatography
● Gas-liquid Chromatography
● Gas-solid Chromatography
Gel Filtration
Gel Filtration (cont’d)
Affinity Chromatography

a method of separating based on a highly specific macromolecular

binding interaction between the biomolecule and another
substance

The specific type of binding interaction depends on the

biomolecule of interest example:
• antigen and antibody,
• enzyme and substrate,
• receptor and ligand,
the ligand is attached to a
solid, insoluble matrix:
• agarose or
• polyacrylamide

chemically modified to
introduce reactive
functional groups with
which the ligand can
react, forming stable
covalent bonds
Affinity Chromatography
Adsorption Chromatography

• Adsorption is completely different from

absorption.
• In here molecules are adsorb to a surface
however molecules will not become a part of
this section. 
• Adsorption chromatography is based on the
interaction between the solute molecules and
active sites on the stationary phase.
Adsorption Chromatography

• based on the interaction between the solute molecules

and active sites on the stationary phase.
• This attachment or interaction depends on the polarity of
solutes.
• This techniques proves the statement that “polar like
polar”
Adsorption Chromatography
(cont’d)

Solvent reservoir

Filter paper disc

Adsorbent

Resin or stationary phase:

Glass wool  Alumina
 Silica gel
Collecting tube
Adsorption Chromatography
(cont’d)

Prepack colloumn
chromatography
Laboratory scale colloumn chromatography
Ion Exchange Chromatography
Reversible exchange of ions in the solution with ions
electrostatically bound to insoluble matrix or stationary phase

separates ions and polar molecules based on their affinity to

the ion exchanger.

Laboratory scale Commercial colloumn Modern ion exchange

chromatography
Ion Exchange Chromatography

– +

– + – – +
+ + –
+ – – +
+ –
+ – +
– – +

ANION exchanger CATION exchanger

with exchangeable with exchangeable
counter ions counter ions
Ion Exchange Chromatography
+
Bounded interest of Ion can be eluted by two
+ – – +
– ways:
– –+
+ +

a. Replace the ion of interest with stronger / higher

magnitude of charge -- so the ion of interest will eluted
out
b. Change the pH of solvent / mobile phase so the
molecule of interest become neutral  eluted

Resin: polysterene , cellulose

Ion Exchange Chromatography (cont’d)
Partition Chromatography
Two kinds of Partition Chromatography:
● Paper Chromatography
● Thin-layer Chromatography (TLC)

Partition Chromatography  for the compounds that are

soluble in both water and organic solvents

Adsorption Chromatography  for the compounds that

are readily soluble in organic liquid but sparingly soluble in
water

Ion Exchange Chromatography  for ionizable water

soluble compounds
Paper Chromatography
Based on direction of solvent flow:
● Ascending Chromatography
● Descending Chromatography
● Circular Chromatography

Distance of migration of X
Rf =
Distance moved by the solvent
Ascending Chromatography
Descending Chromatography
Circular Chromatography
Thin-layer Chromatography
● This method is very rapid (many separations can be
completed under an hour)
● The spots are very compact (so it is possible to
detect compounds at low concentration)
● Compounds separation is much better than paper
chromatography
● Separated compounds can be detected using
corrosive sprays at high temperature
Thin-layer Chromatography
(cont’d)
Thin-layer Chromatography
(cont’d)
Alkaloid using Triterpen & steroid
dragendorf

Phenolic + FeCl3
Two Dimensional
Thin-layer Chromatography
 2D separation
The 1D-TLC separation of
Thymi Oleum compounds. The
mobile phase: toluene and
ethyl acetate (97.5 : 2.5 v/v).
Disemprot menggunakan
vanillin dalam ethanol .
Gas Chromatography
Gas Chromatography (cont’d)
● This method was first described by James and Martein
(1952), and has been developed very rapidly

● This is the best method for separation of biological

compounds

● Advantages of GC:
● Very good separation
● Time (analysis is short)
● Small sample is needed (picogram)
● Good detection system
● Quantitatively analyzed
Principles

(gas)
MOBILE PHASE

Sample Sample
in out

STATIONARY PHASE
(solid or heavy liquid coated onto a solid or support system)
Gas Chromatography (cont’d)
Gas chromatography (GC) is a preferred method,
only applicable to volatile substances

In GC  mobile phase is gas, stationary phase could

be:
Solid  Gas Solid Chomatography (GSC)
Liquid  Gas Liquid Chomatography (GLC)
In GLC, solid support is coated a liquid
Gas Chromatography (cont’d)
Gas chromatography (GC) consists essentially of a
gas supply, column and detector
 Retention Time/Index
• Retention time (RT) is the time taken by an
analyte to pass through a column
• RT is affected by compound, column (dimensions
and stationary phase), flow rate, pressure, carrier,
temp.
• Comparing RT from a standard sample to an
unknown allows compound ID
Gas Chromatography (cont’d)
● Gas supply consist of:
● Cylinder of high purity gas under high pressure
● Gas could be nitrogen, helium, etc.
● Pressure regulation device
● Flow regulation device
● Flow measuring device

● Column:
● Glass/stainless steel
● Containing solid support (GSC)
● Solid support is coated a liquid (GLC)
Gas Chromatography (cont’d)
● Detector is some device which generates a
change in electrical signal in response to the
solute as it comes off the column
● Most detectors require electronic amplification
of the signal (electrometer)
● Kinds of detectors:
● Flame ionization detector (FID)
● Nitrogen phosphorus detector (NPD)
● Electron capture detector (ECD)
● Flame photometric detector (FPD)
Schematic Diagram of
Gas Chromatography
Instrumentation
● Injection port  sample introduction
● Manual - Direct Injection
● Automated - Autosampler
Instrumentation (cont’d)
● Oven  Temperature Control
● Isothermal
● Gradient

240

200

Temp (deg C)
160

120

80

40

0
0 10 20 30 40 50 60
Time (min)
Instrumentation (cont’d)
● Column

Packed

Capillary
Gas Chromatography
GC-Columns

Polysiloxane
Instrumentation (cont’d)
● Detector
● Destructive
● Mass Spectral (CI/EI)
● Flame Ionization (FID)
● Nitrogen-Phosphorus (NPD)
● Flame Photometric (FPD)
● Electrolytic Conductivity (Hall/ELCD)

● Non-destructive
● Thermal Conductivity (TCD)
● Electron Capture (ECD)
● Photo Ionization (PID)
Application of GC and TLC
Application of GC and TLC
(cont’d)
Advantages of LC compared to GC:
1.) LC can be applied to the separation of any
compound that is soluble in a liquid phase.
LC more useful in the separation of biological
compounds, synthetic or natural polymers, and
inorganic compounds

2.) Liquid mobile phase allows LC to be used at lower

temperatures than required by GC
LC better suited than GC for separating compounds
that may be thermally labile
3.) Retention of solutes in LC depend on their
interaction with both the mobile phase and
stationary phase.
•GC retention based on volatility and
interaction with stationary phase
•LC is more flexible in optimizing separations
 change either stationary or mobile
phase
4.) Most LC detectors are non-destructive
•most GC detectors are destructive
•LC is better suited for preparative or process-
scale separations

Disadvantage of LC compared to GC:

1.) LC is subject to greater peak or band-
broadening.
much larger diffusion coefficients of solutes in
gases vs. liquids
 Liquid Chromatography
Classification of Types
• Classification based on pressure:
– Low pressure (gravity based, preparative)
– Moderate pressure (pressure from compressed gases in “flash
chromatography” or from low pressure pumps, also mainly
preparative)
– High pressure (high performance or HPLC)
– Ultra-high pressure (UPLC)

• Classification based on separation mechanism:

– Normal-phase (polar stationary phase, less polar mobile phase)
– Reversed-phase (non-polar stationary phase, more polar mobile
phase)
– Size exclusion chromatography (separation of molecules based
on size)
• The term “Reverse Phase Chromatography”was used
because RP is a form of partition chromatography where
chemically bonded phase is hydrophobic or non-polar
(e.g. octadecyl group), and the starting mobile phase (e.g.
water) must be more polar than the stationary phase.

• This is “reversed” from normal phase chromatography

where the stationary phase is polar or hydrophilic and
the starting mobile phase is more non-polar or
hydrophobic than the stationary phase, hence the term
“Reverse Phase Chromatography”.
… Terima kasih …