Chromatography

T. Trimpe 2006 http://sciencespot.net/

 What is chromatography?
From Wikipedia ...
Chromatography (from Greek word for chromos for colour) is the collective
term for a family of laboratory techniques for the separation of mixtures. It
involves passing a mixture which contains the analyte through a stationary phase,
which separates it from other molecules in the mixture and allows it to be isolated.

Which means ...

Chromatography is the physical separation of a mixture into its individual
components.

We can use chromatography to separate the

components of inks and dyes, such as those found
in pens, markers, clothing, and even candy shells.
Chromatography can also be used to separate the
colored pigments in plants or used to determine the
chemical composition of many substances. http://members.shaw.ca/vict/chemistry_test3.htm
 Chromatography
Derived from the Greek word “Chroma” meaning colour and
“graphy” means writing.
• Chromatography is a technique for separating mixtures into
there component in order to analyze ,identify ,purify and
quantify the mixtures or components

Separate Analyze
Identify
Purify
Quantify
Mixture
Components
 Uses for
Chromatography
Chromatography is used by scientists to:

• Analyze – examine a mixture, its components,

and their relations to one another
• Identify – determine the identity of a mixture or
components based on known components
• Purify – separate components in order to isolate
one of interest for further study
• Quantify – determine the amount of the a mixture
and/or the components present in the sample
 Chromatography
• Chromatography separate molecules using partitioning
characteristics of molecule to remain in a stationary phase versus
a mobile phase
Brief History of Chromatography
• 1903 – Tswett, a Russian botanist
coined the term chromatography.
He passed plant tissue extracts
through a chalk column to
separate pigments by differential
adsorption chromatogrpahy
• 1931 Richard Kuhn used
chromatography to separate
isomers of polyene pigments; this
is the first known acceptance of
chromatographic methods

http://www.chemgeo.uni-hd.de/texte/kuhn.html
 Chromatography
Micheal Tswett invented the chromatography in 1901 during his research on
plant pigments.
He used the technique to separate various plant pigments such as
chlorophyll, xanthophylls, Carotenoids.
 History of the Main
techniques
• 1938 Thin Layer chromatography by Russian
scientist N.A Izamailov and M.S Shraiber
• 1941 Liquid-Liquid partition chromatography
developed by Archer John, Porter Martin
and Richard Laurence Millington Synge
• 1944 Paper Chromatography one of the most
important methods in the development of
biotechnology
• 1945 Gas Chromatography 1st analytical gas-
solid (adsorption) chromatography developed
by Fritz Prior
• 1950 Gas Liquid Chromatography by Martin
and Anthony James; Martin won the Nobel
Prize in 1952
British chemist Archer John Porter Martin, co-
recipient, with Richard L. M. Synge, of the 1952
Nobel Prize in chemistry, "for their invention of
partition chromatography."
 History of the Main
Techniques
• 1966 HPLC named by Csaba
Horvath, but didn’t become a
popular method until 1970s
• 1950s Ion-Exchange
chromatography declassified this
technique
• 1930s Affinity Chromatography
was developed for the study of
enzymes and other proteins

library.thinkquest.org
Examples of Chromatography
Liquid Chromatography
Used to identify unknown plant
pigments & other compounds.

Thin-Layer Chromatography
Uses thin plastic or glass trays to
identify the composition of pigments,
chemicals, and other unknown
substances.

Gas Chromatography
Used to determine the chemical composition of
unknown substances, such as the different
compounds in gasoline shown by each separate
peak in the graph below.
Paper Chromatography
Can be used to separate the
components of inks, dyes, plant
compounds (chlorophyll), make-up,
and many other substances
 Terminology
• Elution • Stationary Phase
- washing of the mixture -
• Eluent • Mobile Phase
- additional solvents used for
elution - fluid carrying the mixture
of analytes
• Effluent
- exiting fluid stream

• Residency
- time spent on column
 Paper Chromatography Lab
• Obtain the supplies you’ll need. Tape – Label with marker
– 1 large beaker (or plastic cup) Pencil
– 1 small beaker (or plastic cup) filled with water
Filter
– 4 pieces of filter paper Paper
– 4 black markers for testing
– 4 small pieces of masking tape
– Pencil (to attach to the top of the filter paper) Ink
– Permanent marker Mark
– Timer
• Write the pen number on a piece of masking tape with a permanent marker
and place it at the top of the strip.
• Choose one of the testing markers and draw a thick line near the bottom of the
filter paper - about ¼ inch from the bottom.
• Pour a small amount of water into the large cup and then hang the paper strip
in the cup. Make sure the ink line does not touch the water – only the bottom
of the filter paper.
• Allow the water to move up the paper for 5 minutes and then remove the strip
from the water. Hang it on the side of the table to dry.
• Follow these directions to test the other pens.
 Illustration of
Chromatography
Stationary Phase

Separation

Mobile Phase

Mixture Components
Component Affinity to Stationary Affinity to Mobile
s Phase Phase
Insoluble in Mobile
Blue ----------------
Phase

Black  

Red  

Yellow          
 Principles of Paper
Chromatography
• Capillary Action – the movement of liquid within the spaces
of a porous material due to the forces of adhesion, cohesion,
and surface tension. The liquid is able to move up the filter
paper because its attraction to itself is stronger than the
force of gravity.

• Solubility – the degree to which a material (solute) dissolves

into a solvent. Solutes dissolve into solvents that have similar
properties. (Like dissolves like) This allows different solutes
to be separated by different combinations of solvents.

Separation of components depends on both their solubility in

the mobile phase and their differential affinity to the mobile
phase and the stationary phase.
Paper Chromatography
Experiment
 Preparing the Isopropanol
Solutions
• Prepare 15 ml of the following isopropanol solutions
in
appropriately labeled beakers:
- 0%, 5%, 10%, 20%, 50%, and 100%
 Preparing the
Chromatography Strips

• Cut 6 strips of filter

paper
• Draw a line 1 cm above
the bottom edge of the
strip with the pencil
• Label each strip with its
corresponding solution
• Place a spot from each
pen on your starting line
 Developing the
Chromatograms
• Place the strips in the beakers
• Make sure the solution does
not come above your start line
• Keep the beakers covered
• Let strips develop until the
ascending solution front is
about 2 cm from the top of
the strip
• Remove the strips and let
them dry
Developing the
Chromatograms
Developing the
Chromatograms
 Observing the
Chromatograms

0% 20% 50% 70% 100%

Concentration of Isopropanol
 Black Dye
1. Dyes separated – purple and black
2. Not soluble in low concentrations
of isopropanol
3. Partially soluble in concentrations
of isopropanol >20%

0% 20% 50% 70% 100%

Concentration of Isopropanol
 Blue Dye
1. Dye separated – blue
2. Not very soluble in low
concentrations of isopropanol
3. Completely soluble in high
concentrations of isopropanol

0% 20% 50% 70% 100%

Concentration of Isopropanol
 Green Dye
1. Dye separated – blue and yellow
2. Blue – Soluble in concentrations
of isopropanol >20%
3. Yellow – Soluble in concentrations
of isopropanol >0%

0% 20% 50% 70% 100%

Concentration of Isopropanol
Alternative Experiments
Alternative Experiments
Alternative Experiments
 THIN LAYER
CHROMATOGRAPHY
Thin layer chromatography is done exactly as it says – using a thin,
uniform layer of silica gel. The silica gel is the stationary phase.
The mobile phase is a suitable liquid solvent or mixture of solvents.

Silica gel is a form of silicon

dioxide (silica). The silicon
atoms are joined via oxygen
atoms in a giant covalent
structure. However, at the
surface of the silica gel, the
silicon atoms are attached to -
OH groups.
 Plate preparation
• Mix the absorbent :- water & Silica gel

• Spread a thin layer of absorbent on an unreactive

hard surface
– Glass, plastic, thick aluminium.

• Heat in oven at 110°C for 30 minutes to activate and

dry the plate
 TLC Procedure
• Place a small amount of
solvent in a beaker

• In pencil, draw a
straight line across the
plate about 1 cm from
the end of the plate

• Place a drop of sample

solution on the line

BEFORE
 Visualization
• Destructive visualization
– Spray plate with H2SO4, and then bake in the oven
at 110ºC for 15-20 minutes. Compound is
destroyed but all spots will be visible

• Non-destructive visualization
– By using UV light
TLC Procedure
 Application

Used in the quantization of plasma Lipids and Fatty

Acids:
Neutral lipids and free fatty acids were extracted
from plasma and separated on TLC plates.
 Column Chromatography
The stationary phase (column packing) in the column is
very polar!

Polar compounds are going to be attracted to the polar

column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly!

Non-polar compounds are going to come off the column

first, while the polar compounds are going to come off
the column last.

Usually, one starts will a less polar solvent to remove

the less polar compounds, and then you slowly
increase the polarity of the solvent to remove the more
polar compounds.
Principles of Separation on
a column
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
 REMEMBER…
• The stationary phase is POLAR
• The more polar component interacts
more strongly with the stationary phase
• The more polar component moves more
slowly.
• The non-polar component moves more
rapidly.
Reverse phase chromatography
Silica is alkylated with long chain hydrocarbon groups, using 18
carbons long. This is usually referred to as C-18 silica.

CH3

CH2
CH3 17 CH3
Si
CH2 SiCH3)3 CH3
17 CH3
SiCH3)3
SiCH3)3
O
Si O
CH3 O
O
O Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si
Si O
Si O
Si O O
Si O O
O O
O O
O
Si
Si O
O
O O
O
 Reverse Phase column
chromatography
• The stationary phase (column packing) is now
NON-POLAR
• Non-polar compounds will move more slowly
because they are attracted to the column
packing.
• The more polar component moves more quickly
down the column.
• Polar solvents, such as water and methanol
are used in reverse phase chromatography
• Used mainly in columns, such as HPLC
HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
(HPLC)
 PRINCIPLE

High performance liquid chromatography is basically a

highly improved form of column chromatography.
Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high
pressures of up to 400 atmospheres. That makes it much
faster .
HOW DOES HPLC
WORK?
 HPLC system
• Solvent Reservoir

• Degasser

• Solvent Delivery System

(Pump)

• Injector

• Column &oven

• Detectors
• Recorder (Data Collection)
 Components
• Solvent Reservoir
– Usually one or more glass or stainless steel reservoirs
each of which contains 200-1000 ml of solvent
• Isocratic elution - single solvent separation technique
• Gradient elution - 2 or more solvents, varied during
separation
 Gradient system
• Isocratic system
– Fixed (un-changeable) mixing ratio during analysis
• Gradient system
– Changeable mixing ratio during analysis
• HPGE (High Pressure Gradient)
• LPGE (Low Pressure Gradient)
 Aim of gradient
- problems in isocratic mode -
• in isocratic mode
Methanol / water = 6 / 4
Long analysis time

Methanol / water = 8 / 2
Poor separations

(Column : ODS type)

 Aim of gradient - solution -

• Gradual change of the mixing ratio during

analysis
95%

Methanol concentration
in mobile phase

30%
Short analysis time
&
Excellent separation
 Degasser
• Problems caused by dissolved air(O2, N2)in mobile phase
– Unstable delivery in pump
– Bigger noise and large baseline-drift in detector cell

 In order to avoid causing the problems,

mobile phase should be degassed.
– vacuum pumping systems
– distillation system
– a system for heating and stirring the solvents
– sparging system - bubbles an inert gas of low solubility through
the solvent
 Column

– straight, 15 to
150 cm in
length; 2 to 3
mm i.d.
– packing - silica
gel, alumina,
Celite
 Types of Detector
• UV/Visible
- Fixed wavelength
- variable wavelength
• Photo Diode Array
• Refractive index
• Fluorescence
• Evaporative light scattering
• Conductivity
• Electrochemical
Refractive Index Detector
INJECTION OF THE SAMPLE:
Injection of the sample is entirely automated.
RETENTION TIME:
The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time at
which the sample is injected to the point at which the
display shows a maximum peak height for that
compound. Different compounds have different
Retention times. The retention time will vary depending
on:
• the pressure used
• the exact composition of the solvent
• the nature of the stationary phase (size & compositition)
• the temperature of the column
 INTERPRETATION :
• The output will be
recorded as a series of
peaks.

• Each peak represents a

compound in the
mixture.

• The area under the peak

is proportional to the
amount of any particular
compound which has
passed the detector.
GAS-LIQUID
CHROMATOGRAPHY
Principle
In gas-liquid chromatography, the mobile phase is a gas such as
helium and the stationary phase is a high boiling point liquid
absorbed onto a solid. How fast a particular compound travels
through the machine will depend on how much of its time is
spent moving with the gas as opposed to being attached to the
liquid in some way.
How does GLC work?
 Retention time
“The time taken for a particular compound to travel
through the column to the detector is known as its
retention time.”
• compounds have different retention times depends
upon:
– The boiling point of the compound.
– The solubility in the liquid phase.
– The temperature of the column.
 INTERPRETATION :
• The output will be
recorded as a series of
peaks.

• Each peak represents a

compound in the
mixture.

• The area under the peak

is proportional to the
amount of any particular
compound which has
passed the detector.
 AREA OF THE PEAK

• If two different substance

has passed through.

• Compound absorbs less

UV light have smaller
area & which absorbs
more UV light have
larger area of its peak.
Study Overview
Conclusion
Intensive Insulin Therapy in
Surgical ICU
 Ion-Exchange
Chromatography
• Usually employed with HPLC
• Ions are charged molecules
- cation positively charged ion
- anion negatively charged ion
• These ions do not separate smoothly under the traditional methods
of the liquid and mobile phases of chromatography
• Requires alteration methods of either the mobile phase or
stationary phase are required
- mobile phase suppresses the ionic nature of the analyte
- stationary phase incorporate ions of the opposite charge to
attract and
retain analyte
 How do you get those
columns to work
• There is a glass column
coated with a resin
polymer
• The resin is either
positively charged (an
acid) or negatively
charged (a base)
• An analyte will have ions
opposite of the resins
charge eluting off the ion
of interest
 Resin
• Common resins are copolymerized styrenes
• vinylic and aromatic functional groups
styrene derivative and divinylbenzene

• Creates better stability due to crosslinking of the benzene rings

• Creates a swelling within the polymer affecting the porosity while
taking in the mobile phase liquid
• Aromatic substitution reaction makes these polymers ideal for charged
functional groups
 Cation Exchange resins
• The functional group in cation exchange resins are usually acids

• Sulfonic acids –SO3H (strong acid resin) are added to the resin by
sulfonation reactions
Res-(SO3H) + M+  Res-(SO3M) + H+

• Carboxylic acid –COOH (weak acid resin)

Res-COOH + M+  Res-COOM + H+
• With both the strong and the weak acid exchange sites an acidic
Hydrogen is attached to a functional group chemically bound to the resin

• Cation exchange is good for removing metal ions from an aqueous solution
 Anion Exchange Resins
• The functional groups added to the resin is similar to cation resins
but are basic instead of acidic
• Quaternary ammonium a strong base -- CH2N(CH3)3+OH-

– CH2N(CH3)3+OH- + B-  Res-CH2N(CH3)3+Cl- + OH-

• Polyalky amine a weak base -- NH(-R)2+OH-

NH(-R)2+OH- + B-  Res-NH(-R)2+B- +OH-

 To Affinity and Beyond…
• The rate of ion exchange is controlled by the law of
mass action. At equal concentration the greater
affinity molecule will control the cation exchange
resins in the acid form.
• However if a much higher concentration of strong acid
passed through the greater affinity molecule, such as
sodium, will form the resin, reversing equilibrium and
convert the resin back to an acidic form.
• Generally it is possible to return either ion exchange
resin column to a desired starting form by passing a
large excess of the desired ion at very high
concentration through the resin
 What makes it unique…
• Careful selection of the ionic
composition of the eluent, and
the gradual adjustment of its
strength during elution using
a controlled gradient
• The components of a mixture
of ions can be induced to
separate just as the
components of a mixture
separated by partitionion
chromatography
• The parameters controlling the
relative residence of the
analyte or other eluent ions is
the resin stationary phase or
the ionic solution mobile phase
1) both the relative selectivity
of the resin for the ions and
their relative concentrations in
each phase
2) In ion exchange, selectivity
resides in relative ion-pairing
interaction strengths only in
the stationary phase
Relative Affinity of Ions
• The higher the charge the higher the
affinity
Na+ < Ca2+ < Al3+ and Cl- < SO42-

• The Ion with the greatest size and

charge has the highest affinity
Li+ < Na+ < K+ < Cs+ < Be2+ < Mg2+ < Cu2+ and F- < Cl- < Br- < I-
 Ion Exchange
Chromatography
The parameters controlling the residence
times of analyte or other eluent ions in the
resin stationary phase or ionic solution
mobile phase are
1) both the relative selectivity of the resin
for the ions
2) their relative concentrations in each
phase
 Applications of IEC
• In cell and molecular biology, ion
exchange chromatography is used to
separate different proteins out of an
eluant.
• areas of research such as the
environment, industry, commercial
products of organic molecules without
UV-vis absorption
Gel Filtration Chromatography
 Original Article
Intensive versus Conventional Glucose Control in
Critically Ill Patients
The NICE-SUGAR Study Investigators

N Engl J Med
Volume 360(13):1283-1297
March 26, 2009
• Gel filtration (chromatography), is also
known as molecular sieve chromatography.
• Gel filtration chromatography separates
molecules according to their size and
shape.
• The stationary phase consists of beads
containing pores that span a relatively
narrow size range.
• Smaller molecules spend more time
inside the beads than larger
molecules and therefore elute later
(after a larger volume of mobile
phase has passed through the
column).
Size exclusion chromatography
Gel filtration chromatography

 Contains porous beads

 Separates according to size and shape
 Larger proteins excluded
from the small pores
 Quaternary structure determination,
& Mr estimation using
a standard curve
(log Mr vs elution volume)

Ⓠ Fibrous proteins
Spherical vs rod-shaped proteins
 Types of gels used
• The gels used as molecular sieves are
cross linked polymers.
• They are uncharged and inert i.e.
don’t bind or react with the materials
being analyzed.
• Three types of gels are used:
 Types of gels cont…
1. Dextran: is a homopolysaccharide of glucose
residues.
• it’s prepared with various degrees of cross-
linking to control pore size.
• It’s bought as dry beads, the beads swell when
water is added.
• The trade name is sephadex.
• It’s mainly used for separation of small
peptides and globular proteins with small to
average molecular mass.
 Types of gels cont…
2. Polyacrylamide:these gels are prepared by cross
linking acrylamide with N,N-methylene bis
acrylamide.
The pore size is determined by the degree of
cross-linking.
The separation properties of polyacrylamide gels
are mainly the same as those of dextrans.
They are sold as bio-gel P. They are available in
wide range of pore sizes.
 Types of gels cont…
3. Agarose: linear polymers of D-galactose and 3,6
anhydro-1-galactose.
It forms a gel that’s held together with H bonds. It’s
dissolved in boiling water and forms a gel when it’s
cold.
The concentration of the material in the gel determines
the pore size.
The pores of agarose gel are much larger than those of
sephadex or bio-gel p.
It’s useful for analysis or separation of large globular
proteins or long linear molecules such as DNA
• The gel filtration material that will be
used in the experiment below is called
Sephadex G-75 and it will separate
molecules with molecular weights from
3,000 to 70,000. Molecules with
molecular weights larger than 70,000
will be excluded from the beads.
 AFFINITY
CHROMATOGRAPHY
 Affinity History
• 1930s, first developed by Arne Wilhelm
Tiselius, won the Nobel Prize in 1948
• Used to study enzymes and other proteins
• Relies on the affinity of various biochemical
compounds with specific properties
ex) enzymes for their substrates
antibodies for their antigens
Affinity chromatography
 Separates by specific interactions
 Contains a ligand:
enzyme ‒ substrate
receptor ‒ hormone
antigen ‒ antibody
(His)6 protein – Ni2+
 So now what….
• The Sample is injected into the equilibrated
affinity chromatography column
• Only the substance with affinity for the ligand
are retained on the column
• The substance with no affinity to the ligand will
elute off
• The substances retained in the column can be
eluted off by changing the pH of salt or organic
solvent concentration of the eluent
 Specificity of Affinity
Chromatography
• Specificity is based on three aspect of
affinity

1) the matrix

2) the ligand

3) the attachment of the ligands to the matrix

 Matrix
• The matrix simply provides a posre structure to increase
the surface area to which the molecule can bind

• This has been what kept the Affinity Chromatography

from being developed earlier and useful to the scientific
community

• The matrix must be activated for the ligand to bind to it

but still able to retain it’s own activation towards the
target molecule
 Matrix
• Amino, hydroxyl, carbonyl and thio groups located
with the matrix serve as ligand binding sites

• Matrix are made up of agarose and other

polysaccharides

• The matrix also must be able to withstand the

decontamination process of rinsing with sodium
hydroxide or urea
 Ligand
• The Ligand binds only to the desired molecule within the solution

• The ligand attaches to the matrix which is made up of an inert

substance

• The ligand should only interact with the desired molecule and form a
temporary bond

• The ligand/molecule complex will remain in the column, eluting

everything else off

• The ligand/molecule complex dissociates by changing the pH

 So many ligands so little
time
• The chosen ligand must bind strongly to the molecule
of interest

• If the ligand can bind to more than onel molecule in

the sample a technique, negative affinity is performed
- this is the removal of all ligands, leaving the
molecule of interest in the column
- done by adding different ligands to bind to the
ligands within the column
 Applications
• Used in Genetic Engineering
• Production of Vaccines
• And Basic Metabolic Research
 Uses for Chromatography
Real-life examples of uses for chromatography:
• Pharmaceutical Company – determine amount of each
chemical found in new product
• Hospital – detect blood or alcohol levels in a patient’s blood
stream
• 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