bringing biotech product to market chapter 9. objectives purifying product define chromatography and...
TRANSCRIPT
Bringing Biotech Product to Market
Chapter 9
Objectives
Purifying product Define chromatography and
distinguish between planar and Column chromatography
Purification Techniques
Column chromatography Sample passed through a
column packed with submicroscopic beads
Beads act as molecular sieves (separate mole. Based on characteristics ex. size, shape, charge)
Column chromatography
Tube with membrane (frit) near the base
Resin beads of particular type (in buffer) are poured into the column
Resin beads settle onto the frit and form matrix
Molec pass through it
Column chromatography
Sample get added to top of resin bed Gravity pulls it into the matrix Depending on the resin, molec will either
bind to beads or pass through them As sample drips out, collected at regular
intervals (fractions) Before sample is passed through column it
must be in the right buffer
Column chromatographyProteins
Since most proteins are colorless their separation on column is not visible
So are fractions collected To visualize which protein separate
into which fraction samples are run on PAGE and ID by MW
By comparing bands in original column load to the bands in the fractions, technicians can determine the amount of separation
Often take more than one kind of column chromatography
ChromatographyTypes
Column chromatography
Planar chromatography Paper Thin layer
Separation method for biomoleculesOutcome of a chromatography experiment is a CHROMATOGRAM
ChromatographyBasics
All chromatographic systems contain: Stationary phase Mobile phase Sample molecules (mixture for separation)
Movement of molecules determined by balance between 2 forces: 1. Mobile phase 2. Stationary phase
ChromatographyBasics
1. Mobile phase (impelling force) Carries with it molecules for which it has affinity -
favored by solubility (LC), volatility (GC) 2. Stationary phase (retarding force)
Holds back molecules with which it interacts Balance between forces differs for different
molecules Different mobilities, separation of components
Column chromatography
Planar Chromatography
Paper
Thin-layer
Column chromatographyTypes
1. Gel filtration (size exclusion) chromatography Sep molec based on size
2. Ion-exchange chromatography Sep molec based on charge
3. Affinity chromatography Sep molec based on shape and unique functional group
4. Hydrophobic- interaction chromatography Sep molec based on hydrophobicity (not soluble in water) Not very common (will not discus further)
Gel filtration (size exclusion) chromatography
Preferred for high weight (>2000-3000) neutral molec Polymers, high MW proteins
Separation b/c solute permeation into solvent filled pores within column packing Large: excluded from pores b/c physical size
No retention Small: permeate greater portion of pores
Retained In between: partial permeation
Retention btw 2 extremes
Ion-exchange chromatography Ion exchanger surface
Cations, anions, water
Cations, or anions chemically bond to insoluble matrix (organic and porous in nature) Chemical bound ions = fixed ions Ions of opposite charge = counter ions Pores have water and sufficient [counter ions] Exchanger electrically neutral
Cations exchanger: fixed ions negative Anion exchanger: fixed ions positive
Counter ions replaced by ions of same charge from external solution
M+E- + A-↔ M+A- + E-
Anion exchanger
Affinity chromatography
Antibody recognizes only certain antigen
Will bind them and pull them out of solution
High performance liquid chromatography HPLC
HPLC
Homework
Sec 9.1 Questions 2 and 3 Sec 9.2 Questions 1, 2, and 3
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