B I O 3 2 1 - B I O P H Y S I C S
CHROMATOGRAPHY
Basic concepts, Principles, Types and Uses
Chavez, Mhark Jelo G. | Delos Santos, Noviel |
Hipolito, Joy D. | Sison, Tracy Millette
2021
CONTENTS
BASIC CONCEPTS OF T Y P E S O F
CHROMATOGRAPHY CHROMATOGRAPHY
PRINCIPLES OF U S E S O F
CHROMATOGRAPHY CHROMATOGRAPHY
BASIC CONCEPTS OF CHROMATOGRAPHY
• a teacher of physics and chemistry at the
university of basel in switzerland in 1828
• He produced over 360 papers in his
lifetime and is credited with the discovery Christian Friedrich Schonbein
(1799-1868)
and naming of ozone in papers published
in 1840.
BASIC CONCEPTS OF CHROMATOGRAPHY
He worked with Schönbein on a
process capillary analysis, and
published over 80 papers related
to capillary analysis.
BASIC CONCEPTS OF CHROMATOGRAPHY
• 1861, Schönbein and Goppelsröder recognized that
when an aqueous dye solution was dripped onto
filter paper and allowed to diffuse, the water
molecules move d fa ste r tha n the dissolve d
components
• They realised that this would allow the components
of the solutions to be analysed.
BASIC CONCEPTS OF CHROMATOGRAPHY
• He developed the concept of liquid
chromatography during his attempt to
purify chlorophylls from plant extracts
• Despite being discovered by Tsvet in
1903, chromatography was largely
ignored for many years.
BASIC CONCEPTS OF CHROMATOGRAPHY
During 1930’s and 1940’s, new types of
chromatography have developed
BASIC CONCEPTS OF CHROMATOGRAPHY
Chromatography, a technique for separating the
components, or solutes of a mixture on the basis of
the relative amounts of each solute distributed
between a moving fluid stream.
BASIC CONCEPTS OF CHROMATOGRAPHY
Chromatograph, equipment that enables a
sophisticated separation of gas chromatography or
liquid chromatography.
Eluent, fluid entering column/ solvent that carries
the analyte.
Eluate, mobile phase leaving the column.
BASIC CONCEPTS OF CHROMATOGRAPHY
Stationary phase, immobilized phase (immobilized
on the support particles or on the inner wall of the
column tubing.
Mobile phase, moves in a definite direction; moves
through the chromatography column (stationary phase)
where the sample interacts with the stationary phase
and is separated.
BASIC CONCEPTS OF CHROMATOGRAPHY
Retention Time, time takes for a particular analyte to pass
through the system under set condition.
Sample, substance analyzed in chromatography
Solvent, any substance capable of solubilizing another
substance
PRINCIPLES OF CHROMATOGRAPHY
Chromatography is based on the principle where molecules in mixture
applied onto the surface or into the solid, and fluid stationary phase is
separating from each other while moving with the aid of a mobile phase.
The various factors working on this process of separation include
molecular characteristics associated with adsorption (solid-
liquid), partition (solid-liquid), and differences or affinity among
their molecular weights.
PRINCIPLES OF CHROMATOGRAPHY
Thus, due to these differences, few components of the
mixture stay for longer period in the stationary phase,
and then move slowly in the chromatography system.
However, other components leave the
chromatography system faster after rapidly passing
into the mobile phase.
Each substance have different
affinity to different substance
Solubility Charge
Chemical
Affinity
PRINCIPLES OF CHROMATOGRAPHY
During the movement of the sample, a
separated result is formed by the repeated
desorption and sorption in the direction of the
mobile phase migration.
An interaction between the molecules are physical and involves weak
chemical bonds like dipole-dipole interaction and hydrogen bond
formation and adhere to the stationary components.
Components that adhere strongly to the stationary phase moves slowly
than those who adhere weakly.
Two Phases of Chromatography
Mobile Phase Stationary Phase
Solvent moving through Immobile or fixed in one
the stationary phase place
Composed of “liquid” or This phase is always
a “gaseous component” composed of a “solid”
phase or “a layer of a
liquid adsorbed on the
surface solid support”.
PARTS OF A
CHROMATOGRAPHY
FINISH LINE
STATIONARY PHASE (CHROMATOGRPHY PAPER)
BEAKER
STARTING LINE
LIQUID LEVEL
MOBILE PHASE
How does it work? S
T
A
T
I
O
N
A
R
Y
P
H
A
S
E
Mobile Phase
How does it work? Retention factor:
Rf=
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TYPES OF CHROMATOGRAPHY
Paper chromatography
Thin-layer chromatography
Column chromatography
Ion exchange chromatography
Affinity chromatography
High performance liquid chromatography
Gas chromatography
Gel permeatin cromatography
TYPES OF CHROMATOGRAPHY
p h y , se p a r a t i o n o f t h e
c h r o m a t o g r a
In a paper a
a p a p e r s t r i p w h i c h i s
r f o r m e d o n
mixture is pe a
l i q u i d so l v e n t a c t s a s
h a s e a n d a
stationary p
mobile phase.
TYPES OF CHROMATOGRAPHY
TYPES OF CHROMATOGRAPHY
• Paper adsorption chromatography- Stationary and
mobile phase molecules act base on degree of interaction
• Paper partition chromatography - moisture acts as
stationary phase while mobile phase molecules is
absorbed.
TYPES OF CHROMATOGRAPHY
w o r k s o n t h e b a s i s o f
r o m a t o g r a p h y
Thin layer ch n a
w h e r e s i l i c a o r a l u m i
solid-liquid p a r t i t i o n
p h a se a n d l i q u i d a s a
s a s t a t i o n a r y
plate used i
mobile phase.
TYPES OF CHROMATOGRAPHY
TYPES OF CHROMATOGRAPHY
• A thin layer of silica gel or cellulose is used as an
adsorbent and the solvent mixture as a mobile phase.
• The mixture of the solution is placed at a distance of 2cm
above one end of the plate. Then the plate is placed in a
jar containing a fluid solvent.
TYPES OF CHROMATOGRAPHY
• Moving phase is drawn up by the capillary action where
the mixture rises up the plate carrying different
components.
• The plate is taken out after the mobile solvent reaches the
top and its retention factor is calculated.
TYPES OF CHROMATOGRAPHY
r a p h y i s a p r e p a r a t i v e
Colu m n C h r o m a t o g
o m p o u n d s d e p e n d i n g
e d t o p u r i f y c
technique us
o r h y d r o p h o b i c i t y
on their polarity
TYPES OF CHROMATOGRAPHY
• Liquid chromatography (LC) - a technique used to
separate when a molecules or ions are dissolved in a
solvent, it separate the components using a column of
adsorbent packed glass tube.
TYPES OF CHROMATOGRAPHY
• Gas chromatography (GC) - a technique of separation
of chemical components which are usually organic
molecules or gases and determines their presence.
TYPES OF CHROMATOGRAPHY
• Stationary phase is packed into a glass or metal column.
• Eluent is passed throught the column by a pumping system
• it can be coated in small particles packed in to the column
or be applied as thin film inside the wall.
TYPES OF CHROMATOGRAPHY
• Adsorption chromatography
• Partition chromatography
• Ion exchange chromatography
• Gel chromatography
TYPES OF CHROMATOGRAPHY
• As the eluent flows through the column the analytes
separate on the basis of their distribution coefficients and
emerge individually in the eluate as it leaves the column.
TYPES OF CHROMATOGRAPHY
a p h y i s t h e s e p a r a t i o n
e c h r o m a t o g r
Ion exchang
o l e c u l e s w h e r e s o l i d
e n t h e c h a rg e d m
betwe
a n g e r e s i n s a r e u s e d .
poly m e r i c i o n e x c h
TYPES OF CHROMATOGRAPHY
• In a column, charged ion resins are packed which is taken
as stationary phase. The mixture with the charged particles
when passed through the column binds to the oppositely
charged resins.
TYPES OF CHROMATOGRAPHY
• If a negative charged molecule is used, it binds to the
positively charged resins and vice versa.
• Then an appropriate buffer is used to separate the complex
charged molecules and resins..
TYPES OF CHROMATOGRAPHY
p h y i s a t y p e o f l i q u i d
Affin i t y c h r o m a t o g r a
p a r a t i o n , p u r i f i c a t i o n
p h y f o r t h e s e
chromatogra .
f s a m p l e c o m p o n e n t s
or specific a n a l y s i s o
TYPES OF CHROMATOGRAPHY
• Agarose or cellulose is loaded in a column as a solid
support. The mixture of mobile phase is placed at a
constant rate.
TYPES OF CHROMATOGRAPHY
• The ligand molecule complex is formed during the process
and rests of the components are eluted out with the mobile
phase.
• Components with the stationary phase are obtained by
changing pH and its ionic strength.
TYPES OF CHROMATOGRAPHY
si o n o f l i q u i d c o l u m n
m o d i f i e d v e r
HPLC is a 0
a l l e r p a r t i c l e s s i z e < 1
a p h y . A s s m
chromatogr
w i t h a h i g h p r e ss u r e
m i c ro n s a r e s e p a r a t e d
TYPES OF CHROMATOGRAPHY
• Cellulose or silica is taken in a column of glass tube where
sample mixture of mobile phase is introduced from the top
of the column and a high pressure is passed through a
pump at a constant rate.
• The components are separated on the basis of different
adsorption.
TYPES OF CHROMATOGRAPHY
• High affinity molecules remain adsorbed for a long time
and moves slowly while low affinity molecules move
faster.
• The process is similar as of liquid column chromatography
but only a solvent is forced under high pressure up to
400atm.
TYPES OF CHROMATOGRAPHY
c h n i q u e o f s e p a r a t i o n
o g r a p h y i s a t e
Gas chromat y
e n t s w h i c h a r e u s u a l l
of ch e m i c a l c o m p o n
a n d d e t e r m i n e s t h e i r
c u l e s o r g a se s
organic mole
presence.
TYPES OF CHROMATOGRAPHY
• It is different from other chromatography because the gas
acts as a mobile phase and the separation is vapor.
• The sample is injected into the column which is either gas
or liquid.
TYPES OF CHROMATOGRAPHY
• Helium is usually used as a carrier gas as a mobile phase
which moves in the analyte through the column at their
own rate.
• The components are collected and their retention time is
determined.
TYPES OF CHROMATOGRAPHY
t o g r a p h y i s a m e t h o d
e a t i o n c h r o m a
Gel perm i n
s o l u t i o n a r e se p a r a t e d
u l e s i n a
where the molec n a s
e i g h t . I t i s a l so k n o w
e i r s i z e o r w
terms of th a p h y
l a r s i e v e c h r o m a t o g r
m o l e c u
gel filtration, and
TYPES OF CHROMATOGRAPHY
• The semi permeable porous polymer gel bead is a
stationary phase molecules that is filled in a column and the
sample is mixed with mobile phase liquid.
TYPES OF CHROMATOGRAPHY
• The molecules are partitioned on the basis of their
respective sizes. Molecules with small size moves fast out
of the column while the large size molecules stay at the
top.
TYPES OF CHROMATOGRAPHY
• If the aqueous solution is used in mobile phase then it is
gel filtration. Similarly, if an organic solvent is used, it is
gel permeation chromatography.
USES OF CHROMATOGRAPHY
r m a c e u t i c a l p r o d u c t s
o f v a r i o u s p h a
detects purity l i k e
n V A R I O U S s a m p l e s
m i n a t i o n i
detects conta
d a n d b e v e r a g e s
in foo
USES OF CHROMATOGRAPHY
i o n o f i m p u r i t i e s f r o m
Used f o r t h e se p a r a t
i n d u s t r i a l p r o d u c t s
various
t u r e s i n c h e m i c a l l a b s
r e a c t i o n m i x
Analysis of
USES OF CHROMATOGRAPHY
u b s t a n c e s p r e s e n t i n a
F Y d i ff e r e n t s
TO IDENTI
mixture
o f f i b e r s i n f o r e n si c s
He l p s i n t h e a n a l y si s
USES OF CHROMATOGRAPHY
e r e n t p h a r m a c e u t i c a l
a s s a y o f d i ff
Allows the
products
m e d i c i n a l p l a n t s a n d
e n t i f i c a t i o n o f
Aids in the id
their composition
USES OF CHROMATOGRAPHY
t i o n o f i m p u r i t i e s a n d
T H E s e p a r a
USED FOR
s b i o l o g i c a l m i x t u r e s
purific a t i o n o f v a r i o u
a n d m e t a b o l i t e s f r o m
i v e m o l e c u l e s
isolation of act
various samples
s i n c r u d e e x t r a c t s
detection of drug
USES OF CHROMATOGRAPHY
si t i v e l y c h a r g e d i o n s
o f w a t e r, t h e p o
purification
n s a n d t h e n e g a t i v e l y
repla c e s h y d r o g e n i o
p l a c e s h y d r o x y l i o n s.
cha rg e d i o n s r e
USES OF CHROMATOGRAPHY
o d f o r t h e a n a l y s i s o f
e ff e c t i v e m e t h
Works as an c i d s
y d r o l y si s o f n u c l e i c a
e d a f t e r h
products form
n o f m e t a l s a n d o t h e r
Facil i t a t e s se p a r a t i o
inorg a n i c c o m p o u n d s
USES OF CHROMATOGRAPHY
a r a t i o n t e c h n i q u e f r o m
Used a s a st a p l e s e p
o t h e r p r o t e i n s
enzymes and
e n - a n t i b o d y r e a c t i o n s
e i n v i t r o a n t i g
Applied in th
USES OF CHROMATOGRAPHY
i o n o f c o m p o n e n t s a s
T H E se p a r a t
USED FOR u r e
p u r i t i e s f r o m a m i x t
m o v a l o f i m
well as the re d
t e c t i o n o f m u t a t i o n a n
Can be use d i n t h e d e
i sm s i n n u c l e i c a c i d s .
nucleotide p o l y m o r p h
USES OF CHROMATOGRAPHY
f p o l l u t a n t s p r e se n t i n
Used in th e a n a l y si s o
n m e n t a l s a m p l e s
enviro
u c t p u r i t y a n d q u a l i t y
Main t e n a n c e o f p r o d
n d u s t r i a l p r o d u c t i o n s
V A R I O U S i
control of
USES OF CHROMATOGRAPHY
l o g i c a l m o l e c u l e s l i k e
f d i ff e r e n t b i o
Separation o
s a n d n u c l e i c a c i d s
protein
t e c h n i q u e m a k e s t h e
d s p e e d o f t h i s
The increase
n d m o r e e f f e c t i v e
process faster a
USES OF CHROMATOGRAPHY
e n t r a t i o n o f d i ff e r e n t
Ca l c u l a t i o n o f c o n c
v a r i o u s s a m p l e s
chemicals in
p o l l u t a n t s , o i l s sp i l l s,
a n a l y si s o f a i r
Used in the
and o t h e r s a m p l e s
USES OF CHROMATOGRAPHY
i e n c e - i d e n t i f y i n g a n d
n f o r e n si c s c
Can be used i d i n
o l o g i c a l s a m p l e s f o u n
a r i o u s b i
quantifying v
the crime scene.
USES OF CHROMATOGRAPHY
Gel Permeation chromatography
• One of its principal advantages is that separation can be performed under conditions
specifically designed to maintain the stability and activity of the molecule of interest with
compromising resolution
• The absence of a molecule-matrix binding step also prevents unnecessary damage to
fragile molecules, ensuring that gel-filtration separations generally give high recoveries of
activity
• It has been used successfully in the purification of proteins and peptides from various
sources DUE TO its unique mode of separation
• Used in the separation of various nucleic acids like DNA, RNA, and tRNA and their
constituent bases, adenine, guanine, thymine, cytosine, and uracil
……
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