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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