1. Lecture 2: Introduction to Chromatography and Size- Exclusion Chromatography
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2. What is chromatography
What is chromatography? It is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase, SP) while the other moves (mobile phase, MP or‘eluant’) in a definite direction
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3. Schematic representation of Column Chromatography
At t = 0 we will open the gate and let the analyte into the column
Analyte will be carried by the Mobile phase (Gas or liquid)
Analyte might partition to Stationary phase (Solid or Liquid)
Analyte will be detected by its adsorption of light at the detector
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4. Schematic representation of Column Chromatography
Assume the mobile phase moves in steps
If analyte has no affinity for SP it would move with the mobile phase
Emerge at the detector at time (tM)
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5. Schematic representation of Column Chromatography
If the analyte has affinity for the stationary phase it will be retarded
It will emerge at the detector after “retention time” (tR)
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6. Corrected retention time (t’R)
Unretained peak (tM)
Analyte peak (tR)
Corrected retention time (t’R)
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7. What is “Retention time”
What is “Retention time”? The “Retention time” of a solute is taken as the elapsed time between the time of injection of a solute and the time of elution of the peak maximum of that solute.
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8. What is “Corrected Retention time”
What is “Corrected Retention time”? The “corrected retention” time of a solute is the retention time minus the retention time of a completely unretained solute.
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9. Distribution of the Analyte between two phases An analyte is in equilibrium between the two phases; Amobile <=> Astationary K, partition coefficient; defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase.
Retention factor, k', is often used to describe the migration rate of an analyte on a column. It is also called the capacity factor. The retention factor for analyte A is defined as;
k'A = tR - tM / tM = t’R/tM
Analytes retention factor is less than one, elution is so fast that accurate determination of the retention time is very difficult
High retention factors (greater than 20) mean that elution takes a very long time
Ideally, the retention factor for an analyte is between one and five.
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10. Selectivity Factor (α)
selectivity factor (α), , which describes the separation of two species (A and B) on the column;
α = k'B / k 'A
species A elutes faster than species B
selectivity factor is always greater than one
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11. Resolution is the measure of how well species have been separated
The resolution of two species, A and B, is defined as
                                                   
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12. Efficiency
To increase the efficiency of a system. This is achieved by:
Well-packed columns, evenly packed, neither to tight or too loose, with no air bubbles
Small matrix particle size, bearing in mind that small particle sizes decrease the flow rates, and hence increase the run times;
Even buffer flow.
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14. Concept of Column Plates or Theoretical Plates
A theoretical plate in a separation processes is a hypothetical zone or stage in which two phases, establish an equilibrium with each other.
If the two phases are liquid and vapor for example:
Vapor-Liquid Equilibrium : Rate of evaporation is equal to the rate of condensation
A collection of theoretical plates or stages is referred to as a theoretical tray
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15. Estimating number of theoretical plates (N) from Retention time
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18. Co-Relation between all that was mentioned
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19. Concept of Retention time Partition Co-efficient and retention factor
Points to Remember
Concept of Retention time
Partition Co-efficient and retention factor
Selectivity Factor
Resolution
Efficiency
Plate Theory of Chromatography
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20. Classification
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21. Size-Exclusion Chromatography (SEC) aka
Gel-Permeation Chromatography (GPC)
Gel Filtration
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22. Definition
Type of chromatography that separates molecules according to differences in size or hydrodynamic volume as they pass through a gel filtration medium packed in a column
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23. Basic Facts
Molecules are separated based on their size and shape using a wide array of porous materials aka gel
Each molecule of the gel has a porous structure
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24. Basic Facts
A column of gel granules, equilibrated with appropriate buffer is set up. Molecules of different molecular sizes are eluted through this column. Large molecules will be excluded from the pores of these granules and will pass thorough the interstitial space and eluted first.
Large molecules will appear from the column first, followed by smaller molecules
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25. Totally included –eluted last Totally excluded –eluted first
Porous beads
column
Totally included –eluted last
Partially included
pores
Totally excluded –eluted first
The above animation describes the duration of molecules of different sizes in the column. Small molecules will diffuse into the porous beads due to concentration gradient.
As mobile phase is continuously supplied, concentration gradient is reestablished.
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26. Basic Facts
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27. Three types of gels are commonly used:
Types of gels used
Cross linked polymers
Uncharged and inert i.e. don’t bind or react with the materials being analyzed
Three types of gels are commonly used:
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28. Dextran It is homo-polysaccharide of glucose residues (Glucan)
It is prepared with various degrees of cross-linking to control pore size.
It is 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.
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29. Polyacrylamide
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
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30. 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
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31. Sephadex – Cross Linking Dextran with Epichlorohydrin
Composite Gels
Sephadex – Cross Linking Dextran with Epichlorohydrin
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32. Superdex – Cross Linking Dextran with Agarose
Composite Gels
Superdex – Cross Linking Dextran with Agarose
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33. Composite Gels
Sephacryl – Covalently cross linking allyl-dextran with N,N-methylene bis acrylamide
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45. Size Exclusion chromatography separates on the basis of size
Points to Remember
Size Exclusion chromatography separates on the basis of size
The analyte(s) do not interact chemically with the matrix – (Molecular sieve)
HMW analytes are eluted first, and LMW analytes later
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46. Uses of Gel-Filtration
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47. Separation of Proteins
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48. Isolation and purification of the novel protein - Ohanin
Isolation and purification of the novel protein. A, gel filtration of king cobra venom. Crude venom (200 mg) was loaded onto a Superdex 30 column (Hiload 16/60). The column was pre-equilibrated with 50 mm Tris-HCl (pH 7.4). Proteins were eluted at a flow rate of 1 ml/min in the same buffer. The horizontal solid bar (peak 1b) indicates the fraction containing the protein of interest. B, RP-HPLC of peak 1b from gel filtration. Jupiter C18 semipreparative column was equilibrated with 0.1% (v/v) trifluoroacetic acid. The protein of interest was eluted from the column at a flow rate of 2 ml/min with a gradient of 38–40% B (80% acetonitrile in 0.1% trifluoroacetic acid). The arrow indicates the peak corresponding to the protein of interest. C, ESI/MS of the novel protein. The protein has a molecular mass of 11, ± 0.67 Da as indicated by Biospec Reconstruct spectrum.
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Pung Y F et al. J. Biol. Chem. 2005;280:
©2005 by American Society for Biochemistry and Molecular Biology

49. Study of Complex Formation
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50. Study of Complex Formation
sTfR (30 μg)
sTfR:Tf (30:3) μg
Study of Complex Formation
sTfR:Tf (30:15) μg
sTfR:Tf (30:45) μg
Tf (50 μg)
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51. Determination of Molecular weight
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52. Determination of Molecular weight
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53. Conformational change by SEC
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54. pH-dependent conformational changes of sMR-HA.
pH-dependent conformational changes of sMR-HA. The occurrence of large conformational changes in the MR was tested by gel filtration chromatography under several conditions. The running buffer was 10 mm Tris, 140 mm NaCl, pH 7.4, supplemented with 10 mMCaCl2 at pH 7.4 (A), with EDTA and no CaCl2 (B), with 10 mm mannose (C), at pH 5.4 (D); 20 μl of human serum was used a molecular weight standard (E).
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Boskovic J et al. J. Biol. Chem. 2006;281:
©2006 by American Society for Biochemistry and Molecular Biology

55. Desalting a sample by SEC
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56. Please Go through the papers in the way that was mentioned previously
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