Transcript Thin-Layer Chromatography

Brief History of Chromatography
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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
1915 R.M Willstatter, German Chemist
win Nobel Prize for similar
experiement
1922 L.S Palmer, American scientist
used Tswett’s techniques on various
natural products
1931 Richard Kuhn used
chromatography to separate isomers
of polyene pigments; this is the first
known acceptance of chromatographic
methods
History of the Main techniques
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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, corecipient, with Richard L. M. Synge, of the 1952
Nobel Prize in chemistry, "for their invention of
partition chromatography."
• Liquid Chromatography was first discovered in 1903
by M.S.Tswett.
• In the mid-1950's, the growth of today’s technique of
HPLC was much slower than the GLC.
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In the late 1970's, researchers began to use relatively
short columns with small particles to achieve faster
separations with better efficiencies and lower back
pressures. This method became known as high
performance liquid chromatography and is extensively
used today.
What on Earth did scientist do before
Chromatography?
-Extraction
is based on the difference in solubility material
is grounded, placed with a solvent which
dissolves soluble compounds. A second
extract solvent . The mixture is placed in a
separatory funnel
-Crystallization
also based on the difference of solubility. The
solubility is solved in a fixed volume of solvent.
The purified compound crystallizes as solution
cools, evaporates or diffuses
-Distillilation
separates components based on their volatility
typically via vaporization-condensation
method
Filtration
separate components of a mixture based on
their particle size. Used most often to
separate a liquid from a solid
‫اجزاء کروماتوگرافی‬
‫فازساکن ‪ :‬فازی است که جداسازی بر روی آن انجام می شود‪.‬‬
‫فازمتحرک ‪ :‬فازی است که جداسازی توسط آن انجام می شود‪.‬‬
‫نمونه ‪ :‬شناسایی یا تعیین مقدار‬
‫اصول کروماتوگرافی‬
‫انواع کروماتوگرافی‬
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
Kinds of Chromatography
1. Liquid Column Chromatography
2. Gas Liquid Chromatography
3. Thin-layer Chromatography
Paper Chromatography and Thin Layer Chromatography (TLC)
Column Chromatography
Gas Liquid Chromatography (GLC)
High Performance Liquid Chromatography (HPLC)
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FOUR BASIC LIQUID CHROMATOGRAPHY
The 4 basic liquid chromatography modes are named according to the
mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
CHROMATOGRAPHY
Chromatography basically involves the
separation of mixtures due to differences in
the distribution coefficient (equilibrium
distribution) of sample components between
2 different phases.
One of these phases is a mobile phase and
the other is a stationary phase.
Distribution Coefficient (Equilibrium Distribution )
Definition:
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Different affinity of these 2 components to
stationary phase causes the separation.
Principles of Chromatography
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Chromatography is used when there is a difference in the retention times of
different components
Two types of phases
1) Stationary phase
2) mobile phases
Properties of Chromatographic Properties
1) immiscible stationary and mobile phases
2) an arrangement where a mixture is depositied at one end of the
stationary phase
3) flow of the mobile phase towards the other end of the stationary
phase
4) different rates of partitioning for each component
5) means for visualizing the separation of each component
Paper Chromatography
Paper chromatography. Molecules separate as they move up the paper. The
distance that the molecules travel depends on their size and solubility in the solvent.
Thin Layer and Paper
Chromatography
A solution of a mixture is applied as a
spot/band at the bottom of the plate and
allowed to travel with the solvent up the
Unknown +
plate.
Mixed
standards
standards
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A
B
C
standards
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A+B+C
A+B+C ?
‫كروماتوگرافی‬
‫صفحه ای‬
‫کروماتوگرافی بر روی غشاء نازک )‪(TLC‬‬
‫‪Rf= A/B‬‬
‫‪B‬‬
‫‪A‬‬
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.
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Separation of components depends on both their
solubility in the mobile phase and their differential affinity
to the mobile phase and the stationary phase.
Thin-Layer Chromatography
(TLC)
• TLC involves spotting a dilute solution (1%) of sample
on one end of a small sheet that has been coated with
silica gel (SiO2) or alumina (Al2O3), known as the
stationary adsorbent phase.
• The sheet is placed upright inside a jar in a small pool
of solvent. As the solvent rises up the sheet by
capillary action, the components travel at different
rates based on competing interactions with the mobile
(solvent) and adsorbent phases.
• SiO2 is used for separation of more polar compounds
while
Al2O3
is
compounds.
used
in
the
separation
of
non-polar
Thin-Layer Chromatography
Thin-layer chromatography. Molecules separate as they move through the silica gel. Thinlayer chromatography is used to separate small molecules, such as amino acids.
Thin-Layer Chromatography (TLC)
• A polar solvent will carry a polar compound farther while a nonpolar solvent will carry a non-polar compound farther.
• Rf value is the ratio of the distance the spot travels from the
origin to the distance the solvent travels.
Detectors
• 1) UV lamp
• 2) Iodine
• 3) Specific reagents
TLC
‫آشکارسازها‬
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
Affinity Chromatography.
Separating molecules based
on shape is often done
using antibody resin.
Antibodies recognize only
certain antigens and will
bind those and pull them out
of solution (fraction #3).
Affinity Chromatography
Affinity Chromatography
Surface bound with
Epoxy, aldehyde or aryl ester groups
Metal Interaction Chromatography
Surface bound with
Iminodiacetic acid + Ni2+/Zn2+/Co2+
How do they get those iddy bitty
molecules in there?
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
Applications
• Used in Genetic Engineering
• Production of Vaccines
• And Basic Metabolic Research
Ion-Exchange Chromatography
Ion Exchange Resin.
Resins are manufactured
with ions attached. The ions
present a certain degree of
positive or negative charge,
depending on the buffer pH.
Cation Exchange resins
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The functional group in cation exchange resins are usually acids
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Sulfonic acids –SO3H (strong acid resin) are added to the resin by
sulfonation reactions
Res-(SO3H) + M+  Res-(SO3M) + H+
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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
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Cation exchange is good for removing metal ions from an aqueous solution
Anion Exchange Resins
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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-
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Polyalky amine a weak base -- NH(-R)2+OHNH(-R)2+OH- + B-  Res-NH(-R)2+B- +OH-
MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
pH2
SO3
-
+
Na
H3N
+
COOH
Ion-exchange Resin
SO3
-
H3 N
Na
+
+
-
COO
pH4.5
ION-EXCHANGE CHROMATOGRAPHY
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SO3 Na
+
Separation in Ion-exchange Chromatography is based on the competition of
different ionic compounds of the sample for the active sites on the ionexchange resin (column-packing).
GEL-PERMEATION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical sorting of molecules based on
the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore, retained
longer than large molecules.
Hemoglobin A1c
The HgA1c is the gold standard for diabetes care. To
help prevent diabetes complications, the HgA1c
should be maintained at 7% or below according to the
Diabetes Control and Complications Trial (DCCT) and
United Kingdom Prospective Diabetes Study (UKPDS)
findings.
According to the United Kingdom Prospective
Diabetes Study for every percentage point decrease in
HbA1c (e.g. 9 to 8 percent), there was a 35 percent
reduction in the risk of micro vascular complications.
(ADA, Clinical Practice Recommendations, 2002).
HbA1c: the blood test with a memory
What is HbA1c?
Hemoglobin is a protein that makes
your red blood cells red-colored.
When hemoglobin picks up glucose
from your bloodstream, the hemoglobin
becomes glycosylated.
HbA1c in your bloodstream.
Glycosylated hemoglobin is HbA1c.
The HbA1c test measures the
percentage of HbA1c in your blood—
a number that corresponds to your
average blood glucose for the previous
3 months.
• The hemoglobin A1C percentage is a way of looking at
average blood sugar control over a period of 3 months.
• When plasma glucose is episodically elevated over time,
small amounts of hemoglobin A are nonenzymatically
glycosylated to form HbA1C.
• Red blood cells live 90 to 120 days. This means that
once sugar has combined with the hemoglobin in red
blood cells, the hemoglobin A1C stays in the blood for 90
to 120 days. This means the amount of hemoglobin A1C
in blood reflects how often and how high the blood sugar
has been over the past 3 months.
• The hemoglobin A1C percentage rises as the average
plasma glucose level rises.
Depending on the assay method, HbA1 represent
approximately 3-6 % of total hemoglobin in nodiabetics, and as
much as 20% or more in poorly controlled people with diabetes.
Hemoglobin may become glycated at sites other than the ends
of the beta chains, e.g., at lysine residues (HbA0). These
modifications, which represent almost 50% of the total GHb, do
not alter the charge of the hemoglobin molecule,
thus are not detected by methods based on charge differences
between glycated and non-glycated hemoglobin.
The minor hemoglobin components first recognised
because some of them showed differences in
electrical charge; they were called
"fast haemoglobin"
because they showed less positive charge at neutral
pH, and migrated more rapidly
then hemoglobin A when placed in an electrical field
Hemoglobin Nomenclature
HbA The major form of hemoglobin, a native, unmodified tetramer consisting of two α and two
β chains.
HbA0 The major component of HbA, identified by its chromatographic and electrophoretic
properties. Post translational modifications, including glycosylation, do exist in this fraction but do
not significantly affect the charge properties of the protein.
HbA1 Post translationally modified more negatively charged forms of HbA, as detected by
chromatographic and electrophoretic methods.
HbA1a1, HbA1a2 Chromatographically distinct components of HbA1.HbA1b, HbA1c
HbA1c Adduct of glucose attached to the β-chain terminal valine residue by a ketoamine
linkage.
Pre-HbA1c A labile form of glycosylated hemoglobin containing glucose attached by aldimine
linkage at the β-chain terminal valine residue.
"Fast" The total HbA1 fraction, which, because of a more negative hemoglobin(s) charge,
migrates more rapidly toward the anode in electrophoresis and elutes earlier in cation-exchange
chromatography than does HbA0.
Glycosylated Haemoglobin modified by glucose at α-and β-chain terminal valine residues and
ε-amino groups of lysine residues.
Glycated (glycosylated) haemoglobin(s) A generic term of hemoglobin containing glucose and
(or) other carbohydrate
Terminology
• Elution
- washing of the mixture
• Eluent
- additional solvents used for
elution
• Effluent
- exiting fluid stream
• Residency
- time spent on column
• Stationary Phase
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• Mobile Phase
- fluid carrying the mixture of
analytes
Vocabulary
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Paper chromatography – a form of chromatography that uses filter paper as
the solid phase, and allows molecules to separate based on size or solubility in
a solvent
Thin-layer chromatography – a separation technique that involves the
separation of small molecules as they move through a silica gel
Chromatograph – the medium used in chromatography (ie, paper, resin, etc.)
through which the molecules of interest move and separate
Gel-filtration chromatography – a type of column chromatography that
separates proteins based on their size using size-exclusion beads; also called
size-exclusion chromatography
Ion-exchange chromatography – a separation technique that separates
molecules based on their overall charge at a given pH
Affinity chromatography – a type of column chromatography that separates
proteins based on their shape or attraction to certain types of chromatography
resin
Hydrophobic-interaction chromatography – column chromatography that
separates molecules based on their hydrophobicity (aversion to water)
Elution – when a protein or nucleic acid is released from column
chromatography resin
Cation exchange – a form of ion-exchange chromatography in which positively
charged ions (cations) are removed by a positively charged resin