1. Biophysical Tools '04 - Chromatography
11/15/2018

2. Partition chromatography
redistribution (partition) of the molecules between the stationary and mobile phase
Plate model of chromatography of mixture of compounds partitioning 1:1 (bold) and 1:3 (italics) between the mobile and stationary phase.
larger number of plates – better separation
Biophysical Tools '04 - Chromatography
11/15/2018

3. Biophysical Tools '04 - Chromatography
Adsorption
stationary phases: alumina, activated carbon, hydroxyapatite
Column Support
Application
mesh size
Preparative
50-100
Analytical
high resolution
HPLC
>400
Adsorption
greater surface/volume ration results in larger number of plates (better separation)
flow rate decreases with smaller beads
Biophysical Tools '04 - Chromatography
11/15/2018

4. Biophysical Tools '04 - Chromatography
TLC and GLC
Thin layer chromatography
Gas-liquid chromatography
Thin layer chromatography
v. fine particles, high stationary/mobile phase ratio results in extremely high number of plates
advantages: great resolution, easy extraction
can be used for gel exclusion chromatography
Gas-liquid chrom.
mobile phase is gas (argon), stationary phase is liquid (e.g. PEG) adsorbed to the walls or on the solid support
lipids and small organic molecules: alcohols, amines, esters
sample has to be volatile
good for lipids and small organic molecules: alcohols, amines, esters
much faster than liquid column chromatography
Biophysical Tools '04 - Chromatography
11/15/2018

5. Kinds of chromatography
Biophysical Tools '04 - Chromatography
11/15/2018

6. Biophysical Tools '04 - Chromatography
Gel exclusion
 Matrix
inert beads with pores allowing molecules of certain dimension to enter the beads
large molecules which are to big to enter are eluted in the void volume
small molecules which enter the beads are retarded and elute later
Dextran
Sephadex (< 600 kDa), Sephacryl (x-linked with bis-acrylamide, range 10kDa – 1,500 kDa))
Agarose
larger pores for larger molecules (> 100,000)
beaded form Sepharose, Bio-gel A,
Sepharose CL (x-linked to be resistant to urea, GdCl or temp)
Polyacrylamide
similar to Dextrans with larger range
Bio-gel P
Biophysical Tools '04 - Chromatography
11/15/2018

7. Molecular Weight by Gel Exclusion
Blue dextran for void volume !
Sample volume < 0.5% of the column volume
Biophysical Tools '04 - Chromatography
11/15/2018

8. Biophysical Tools '04 - Chromatography
Optimization
rate
column height
Biophysical Tools '04 - Chromatography
11/15/2018

9. Biophysical Tools '04 - Chromatography
Sample size
Biophysical Tools '04 - Chromatography
11/15/2018

10. Ion-Exchange chromatography
Binding groups
cation exchangers:
sulfonic group SO3- (strong);
phenolic hydroxyl,
carboxyl groups (weak)
anion exchangers: quaternary, ternary amine
Things to watch
Elution with salt, pH
Binding at proper pH
Biophysical Tools '04 - Chromatography
11/15/2018

11. Biophysical Tools '04 - Chromatography
Elution gradient
Keep gradient shallow
salt gradients or pH
Biophysical Tools '04 - Chromatography
11/15/2018

12. Biophysical Tools '04 - Chromatography
pH of the elution
Biophysical Tools '04 - Chromatography
11/15/2018

13. Biophysical Tools '04 - Chromatography
HPLC
Extremely large number of plates  high pressure
rate of elution
For higher resolution need larger number of plates.
Larger number of plates achieved by finer matrix.
Biophysical Tools '04 - Chromatography
11/15/2018

14. Other types of chromatography:
Affinity
(couple ligand to CNBr-activated Sepharose)
Hydrophobic
(low pressure RP-HPLC equivalent)
Affinity chrom
Fish out the desired molecule from a mixture by high affinity “hook”. The “hook”, ligand, can be coenzyme, antibody, inhibitor, anything what binds with high affinity to the molecule.
Couple ligand to matrix by CNBr-activated Sepharose.
Use CH spacers to dangle the ligand away from the matrix. CNBr Sepharose, CH-Sepharose, or AH-Sepharose (carboxyl, amino group at the end respectively) can be coupled with carbodiimide to the ligand.
Popular in antibody purification.
Hydrophobic chrom.
Hydrophobic interactions with octyl-agarose or phenyl-agarose.
Strengthen hydrophobic interaction by increasing polarity of the solvent e.g. 1M ammonium sulfate, elute by decreasing salt or adding EtOH.
Biophysical Tools '04 - Chromatography
11/15/2018

15. Filtration and Dialysis
change buffer often
Reverse dialysis
Filtration Membranes
nitrocellulose
pores 0.01 um – 14 um, adsorbs some proteins, DNA smaller than pores (used in binding assays)
wash with low ionic strength buffers and BSA
cellulose acetate adsorbs less
Centricons
fiberglass
depth filter, larger pores
Dialysis
equilibration of the permeating molecules concentration accros semipermeable membrane
Keep changing buffer
Filtration
nitrocellulose & fiberglass
Centricon series
Biophysical Tools '04 - Chromatography
11/15/2018