Isoelectric focusing electrophoresis- Principle , procedure and applicationsJaskiranKaur72
IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient. The protein becomes “focused” at a point on the gel as it migrates to a zone where the pH of the gel matches the protein's pI. At this point, the charge of the protein becomes zero and its migration ceases.
What is Chromatography?
Applications of Chromatography
Types of Chromatography
1- Column Chromatography
2- Planar chromatography
Paper Chromatography
Gas Chromatography
Detectors
Ion-Pair chromatography is an alternative to ion exchange chromatography.
ion pair reagent.
Mechanism of ion pair chromatography.
Factors influencing retention.
experimental conditions.
Isoelectric focusing electrophoresis- Principle , procedure and applicationsJaskiranKaur72
IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient. The protein becomes “focused” at a point on the gel as it migrates to a zone where the pH of the gel matches the protein's pI. At this point, the charge of the protein becomes zero and its migration ceases.
What is Chromatography?
Applications of Chromatography
Types of Chromatography
1- Column Chromatography
2- Planar chromatography
Paper Chromatography
Gas Chromatography
Detectors
Ion-Pair chromatography is an alternative to ion exchange chromatography.
ion pair reagent.
Mechanism of ion pair chromatography.
Factors influencing retention.
experimental conditions.
High- performance Liquid Chromatography”/
(High- pressure Liquid Chromatography) is a powerful tool in analysis, it yields High Performance and high speed compared to traditional columns chromatography
Dr.S.Karthikumar
Asst. Prof., Dept. of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India
skarthikumar@gmail.com
nanoparticle is part of novel drug delivery system. in this presentation we will study about the concept of nanoparticle, classification of nanoparticle, method of preparation, advantages & disadvantages with marketed formulation.
Stationary Phase and Mobile Phase Selection for Liquid Chromatography
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Principles of chromatography
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As we all know chromatographic fingerprinting of botanicals is a quite recent concept. This presentation will help to the beginners to understand basic thories and fundamantals of thin layer chomatography. The presentation will also provide basic experiemental understanding to perfrom HPTLC fingerprinting of samples/extracts/formulations.
Mixtures of compounds are very common in Organic Chemistry. Most reactions produce more than one product. Naturally occurring materials are only rarely 100% pure. It is therefore desirable to have a simple, fast and efficient way to determine the purity of Organic mixtures. The separation of a mixture by passing it, in solution, over an adsorbent (such as Alumina or Silica Gel) is the basic idea of Chromatography. Chromatography is a very general phenomenon. It involves the passage of a mobile phase across a stationary phase in a column. Usually a mixture of compounds is present in the mobile phase
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2. Chromatography Components
stationary phase (eg., solid matrix)
mobile phase
(eg., solvent) solute
Solutes which interact differently with
the stationary phase can be separated.
4. Common Media Used in Liquid
Protein Chromatography
Media Type Discrimination
Ion Exchange Charge
Gel Filtration Size and Shape
Hydrophobic Surface Hydrophobicity
Reverse Phase Total Hydrophobicity
Affinity Specific Amino Acids
Adsorption Amino Groups?
5. Chromatography
Generic Protocol
1. Prepare Column ()
2. Apply Sample
3. Wash
4. Elute
5. Analyze Fractions
Equipment
• batch-wise
• home made
• work stations
• FPLC/HPLC
HPLC = High Performance (Pressure) Liquid Chromatography
FPLC = Fast Protein Liquid Chromatography
10. Amino a. pKa
Asp, Glu 4.3-4.7
His 7
Lys, Arg > 10
• Prepare or purchase column
• Adjust pH and initial counter ion
• Apply sample
11. Elution from IEC Column
• change pH
• increase counter-ion (ie, salt)
concentration
• in steps (eg, 0.1, 0.2, 0.3, 0.4 M NaCl)
• gradually (eg, 00.4 M NaCl) with
gradient maker
12. • collect fractions as
column elutes
• analyze fractions for
components of interest
13. increasing salting out effect
anions: PO4, SO4, Cl, Br, NO3, Cl04, I, SCN
cations: NH4, Rb, K, Na, Li, Mg, Ca, Ba
increasingchaotropic effect
Hydrophobic Interaction
Chromatography (HIC)
• separates proteins based on differences in
hydrophobicity
• absorb proteins to
hydrophobic matrix
• high salt promotes
hydrophobic interactions
• eg, 1 M (NH4)2SO4
14. HIC vs RPC
Mobile Phase
Polar
Solvent
Nonpolar
Solvent
Conditions Native Denatured
Solute
Discrimination
Surface
Residues
Total
Residues
Reverse Phase Chromatography
• separation based on total hydrophobicity
• generally used to separate small peptides
15. Gel Filtration
• separation based on size, aka
• molecular sieve chromatography
• size exclusion chromatography
• media composed of cross-
linked polymers
• ‘pore’ size of matrix determines
degree of interaction
• larger molecules are excluded and
migrate faster
• smaller molecules are included
and are retained longer
Dextran (=Sephadex®)
Agarose (=Sepharose®)
Polyacrylamide
16.
17. Sephadex
Code Range (kDa)
G-25 1-5
G-50 2-30
G-100 4-150
G-150 5-300
G-200 5-600
• choose matrix with desired characteristics
• size range
• does not interact with solute
• include 0.15-1 M NaCl in buffer
• load sample in smallest possible volume
• elute in one column volume
Practical Considerations
Applications:
• purification
• desalting
• size determination
18. Calculating Size
Vo = void volume
Vt = total volume
Ve = elution volume
Ve - Vo
Vt - Vo
Kav =
• use size standards
• (relative MW)
• migration also
affected by shape
19. Affinity Chromatography
• based on specific binding of
protein to “ligand”
• ligands can include:
• substrate analogs, inhibitors
• natural ligands
• co-factors, metals
• binding proteins
• antibodies
• Elution: destabilize binding
• compete with free ligand
• change pH, ionic strength
• chaotropic or denaturing agents
