Separation of Amino Acids by Gel

filtration chromatography and

Ion Exchange Chromatography

Elizabeth K Laryea-Akrong

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Amino acids
• 20 amino acids that make up
proteins.
• R-group is what brings the
differences

www.google.com
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Amino acids

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Amino acids

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Amino acids

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Formation of peptides
• During amino acid polymerization, the carboxyl group of one amino
acid is linked to the amino group of the next amino acid via a peptide
bond with the loss of a water molecule.

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Structures of proteins

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Chromatography
• Important biophysical technique that enables the separation, identification,
and purification of the components of a mixture for qualitative and
quantitative analysis.
Types :
• Column chromatography
• Ion-exchange chromatography
• Gel-permeation (molecular sieve) chromatography
• Affinity chromatography
• Paper chromatography
• Thin-layer chromatography
• Gas chromatography
• Dye-ligand chromatography
• Hydrophobic interaction chromatography
• High-pressure liquid chromatography (HPLC) 8
TERMINOLOGY
• Elution: The process by which the adsorbed ions are removed from the
column

• Eluent: The solution used for elution

• Eluate: The solution obtained as a result of elution

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 Gel Filtration Chromatography (size exclusion
chromatography)

• NB: Molecules of interest interact differentially with the stationary phase

and a mobile phase and thus can be separated.
• A type of size exclusion chromatography, can be used to separate
molecules and complexes in a sample based on molecular size.
• Materials in solid phase eg: dextran, agarose, poly acrylamide

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 Gel Filtration Chromatography
Principle:
• A resin selected has pores through
which smaller molecules can pass
but larger molecules are excluded
and thus are eluted first.

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Gel Filtration Chromatography

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 Advantages
• Can be used to fractionate molecules and complexes within a
predetermined size range
• Removal of large protein complexes
• Excellent for Removal of small molecules such as nucleotides ,
primers, dyes and contaminant such as salts and unbound detergents,
from a purified protein in preparation for structural or functional
analysis
• Assessment of sample purity
• Can handle biomolecules that are sensitive to changes in pH,
concentration of metal ions or harsh environmental conditions
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 Question?
• A researcher performed gel filtration chromatography for a sample
containing 3 proteins. Protein A- had a molecular weight 10kD, Protein
B- had a molecular weight 50kD and Protein C had a molecular weight
of 100kD. Which protein will elute first from the column and why?

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Ion exchange chromatography
• Separation process
• Separates ions and polar molecules based on their charge
• Principle of separation is by reversible exchange of ions between the
target ions present in the sample solution and their affinity to the ion
exchangers
• Eg: amino acids, peptides, polypeptides, proteins, nucleotides
• 2 types
• The interactions between ions in the sample mixture (protein)and
oppositely charged ligands on the matrix in ion-exchange
chromatography are due to the electrostatic forces
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AEC-Anion Exchange Chromatography-
• Anion exchange chromatography is the
separation technique for negatively
charged molecules by their interaction
with the positively charged stationary
phase in the form of ion-exchange
resin eg:DEAE-cellulose.
• The stationary phase are resins bound to
amine groups or quartenary ammonium
groups
• Strong base exchangers-Quarternary
ammonium.
• Weak base exchangers-Polyalkyl amine

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AEC-Anion Exchange Chromatography-

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 CEC-Cation Exchange Chromatography
• Cation Exchangers
• Positively charged molecules are
attracted to negatively charged solid
support eg: CM-cellulose
• Functional group usually acids-
sulphonic, carboxylic, phenolic groups
with equal amount of cations
• Can be used for also removing metal
ions from solutions
a. Strong cation exchangers-S02H

b. Weak cation exchangers-COOH, OH, SH

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CEC-Cation Exchange Chromatography

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Summary

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Types of resins
• Resin choice is as a result of substances to be separated
• Resins should have small particle size
• Four types of resins can be used in ion exchange chromatography.
• Strongly Acidic Cation Exchange resin
Eg: Sulphonated polystyrene
• Weakly Acidic Cation Exchange resin
Eg: Carboxylic polymethacrylate
• Strongly Basic Anion Exchange resin
Eg: Quarternary Ammonium Compounds
• Weakly Basic Anion Exchange resin
Eg: Phenol formaldehyde and polyamine polystyrene resins

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Preparation of column
• Process of packing is known as wet packing
• Glass wool or cotton plug is placed in the column as a support
• Slurry of resin is made with water
• The fine particles are removed by decantation
• Column is held in vertical position and slurry of resin is then poured into the column
• There should be no air bubble
• Slurry should be added in several parts and allowed to settle between each addition
• Eluent should be allowed to pass through the column
• Level of water should never fall below the surface of the resin
• When sample solution is prepared it is introduced onto the column using a syringe
or micropipette
• Separation can be visualized by
• Spectrophotometry 23
Importance
• It is one of the most efficient methods for the separation of charged
particles.
• It can be used for almost any kind of charged molecule including large
proteins, small nucleotides and amino acids.
• Ion exchange is used for both analytical and preparative purposes in
the laboratory.

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Thank you!!!!

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