Biophysical Tools '04 - Chromatography 11/15/2018

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

Biophysical Tools '04 - Chromatography Adsorption stationary phases: alumina, activated carbon, hydroxyapatite Column Support Application mesh size Preparative 50-100 Analytical 100-200 high resolution 200-400 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

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

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

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

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

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

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

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

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

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

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

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

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