Protein Chromatography

Gel Filtration Chromatography

Gel filtration chromatography seprarates proteins, peptides, and oligonucleotides on the basis of size. Molecules move through a bed of porous beads, diffusing into the beads to greater or lesser degrees. Smaller molecules diffuse further into the pores of the beads and therefore move through the bed more slowly, while larger molecules enter less or not at all and thus move through the bed more quickly. Both molecular weight and three-dimensional shape contribute to the degree of retention. Gel Filtration Chromatography may be used for analysis of molecular size, for separations of components in a mixture, or for salt removal or buffer exchange from a preparation of macromolecules.

Molecular Weight Ranges for Gel Filtration Media



Molecular Weight Ranges for Gel Filtration Media

Molecular Weight Ranges for Gel Filtration Media Image

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Hydrophobic / Lipophilic

Hydroxypropyl beaded dextran (Lipohilic Sephadex®), substituted with long chain alkyl ethers.

Medium Properties / Limitations Potential Applications
Matrix: hydroxyalkoxypropyl-dextan Clean with petroleum ether and acetone Separation fatty acids and esters
Type I: alkyl chain length C11-C14
Type VI: alkyl chain length C15-C18
Type IX: alkyl chain length C15-C18
No decomposition over a six month period of continual use Fractionation of triglyceride mixtures
    Separation of phospholipids, cholesterol and steriods

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Sephadex is a registered trademark of GE Healthcare.

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Sephacryl®

Cross-linked co-polymer of allyl dextran and N,N’-methylenebisacrylamide. The HR grades are smaller particle sizes with narrower size distributions optimized for more efficient separations with faster flow. Gives fast and easy high-resolution results in both lab and industrial scales.

Medium Fraction Ranges (proteins) Properties / Limitations Potential Applications
Matrix: acrylic (dextran/bisacrylamide copolymer) S-100 HR: 1K – 100K Clean with 0.2M NaOH, 0.1M HCl, 1.0M HOAc
Sterilize by autoclave
S-100 HR: peptides, small proteins
25-75 µm
S-1000: 40-105 µm
S-200 HR: 5K – 250K Compatible with detergents, chaotropic salts, dissociation agents S-200 HR: proteins, some small serum proteins
0.2 MPa S-300 HR: 10K – 1500K Compatible with organic solvents S-300 HR: membrane & serum proteins, monoclonal antibodies
pH range: 2-11 S-400 HR: 20K – 8000K Scaleable
Stable bed volume
S-400 HR: large proteins & macromolecules with extended structures (e.g. proteoglycans, liposomes)
  S-500 HR, S-1000: ND Good recoveries S-500 HR: large macromolecules, small particles (e.g. plasmids)

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Sephacryl is a registered trademark of GE Healthcare.

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Sephadex®

Beads are prepared by cross-linking dextan with epichlorohydrin. Widely used in industrial processes. Also useful in desalting and buffer exchange.

Medium Fraction Ranges (proteins) Properties / Limitations Potential Applications
Matrix: dextran (crosslinked with epichlorohydrin) G-10: <700 Clean with 0.2M NaOH
Sterilize by autoclave
G-10: very low MW substances; desalting; buffer exchange; peptides
C: 100–300 µm
M: 50–150 µm
F: 20–80 µm
SF: 10–40 µm
G-15: <1500 Compatible with detergents, chaotropic salts, dissociation agents G-15: low MW compounds; desalting; buffer exchange; peptides
SF: 0.016–0.160 MPa G-25: 1K –5K Interact with aromatic compounds G-25: routine desalting; small peptides & proteins; buffer exchange
pH range:
2–13 (<G-25)
2–10(>G-25)
G-50: 1.5K – 30K Classic Support
Swelling 2 – 40X
G-50: desalting; buffer exchange; standard for process scale; very low nonspecific interactions
  G-75: 3K – 80K C: for very crude samples
M: for standard lab work
F: for preparative lab work
SF: for best resolution, low flow rate
G-100: very low nonspecific interactions
  G-100: 4K – 150K Ideal for MW determinations  

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Sephadex is a registered trademark of GE Healthcare.

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Sepharose®

Beaded agarose for fractionation molecules of high molecular weight. Cross-linked beaded agarose is more resistant to denaturing conditions, and thus offers more versatility in the choice of sample buffer and eluent. The approximate % agarose concentration is indicated by the first number of the Stock Number. Often used as a base matrix for coupling affinity ligands or separation of very large molecules and virus particles.

Medium Fraction Ranges (proteins) Properties / Limitations Potential Applications
Matrix: agarose 6B: 10K – 4000K Clean with 0.5M NaOH
Cannot be autoclaved
Temperature range: 4–40 °C
6B: polio virus purification; proteins; polysaccharides; affinity support
6B: 45 – 165 µm
4B: 45 – 165 µm
2B: 60 – 200 µm
4B: 60K – 20,000K Incompatible with oxidizing agents, chaotropic salts, dissociating agents 4B: tRNAs; membrane proteins; polysccharides; affinity support
0.004 – 0.02 MPa 2B: 70K – 40,000K Broad fraction range 2B: DNA-protein complexes; viruses; asymmetric molecules; affinity support
pH range: 4–9   Good recoveries  

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Sepharose is a registered trademark of GE Healthcare.

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Superdex®

Composite of cross-linked agarose and dextran. High-resolution with short run times and good recovery.

Medium Fraction Ranges (proteins) Properties / Limitations Potential Applications
Matrix: dextran (covalently bonded to agarose) 30: <10K Clean with 1M NaOH, 0.1M HCl, 1.0M HOAc
Sterilize by autoclave
30: peptides; oligosaccharides; small proteins. Selectivity between Sephadex G-25 & G-50
24 – 44 µm 75: 3K – 70K Can be used with detergents, chaotropic salts, dissociating agents 75: high resolution prep separations; wide range of recombinant DNA products. Selectivity similar to Sephadex
G-75
0.5 MPa 200: 10K – 600K Compatible with organic solvents 200: monoclonal antibodies; high resolution prep separations; contaminants of low MW; good when protein MW is unknown. Selectivity similar to Sephadex
G-200
pH range: 3–12   Scaleable
High recoveries
Highest selectivity
 

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Superdex is a registered trademark of GE Healthcare.

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Superose®

Highly cross-linked beaded agarose.

Medium Fraction Ranges (proteins) Properties / Limitations Potential Applications
Matrix: agarose (highly crosslinked) 12: 1K – 300K Clean with 0.2M NaOH, 0.01MHCl, 1.0M HOAc
Sterilize by autoclave
Preparative work: proteins; DNA fragments; polysaccharides
20 – 40 µm 6: 5K – 5000K Compatible with detergents, chaotropic salts, dissociating agents  
12: 0.7 MPa 6: 0.4 MPa Compatible with organic solvents  
pH range: 2–14   Some hydrophobic interactions  

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Superose is a registered trademark of GE Healthcare.

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Toyopearl® HW

Toyopearl is a totally porous, semirigid spherical gel designed for medium and low pressure liquid chromatography. Toyopearl HW gels are synthesized from hydrophilic vinyl polymer containing numberous hydroxyl groups and are composed exclusively of C, H and O atoms. Toyopearl HW is very strong mechanically and can be used at high flow rates.

Medium Fraction Ranges (proteins) Properties / Limitations Potential Applications
Matrix: methacrylate (ethylene glycol/methacrylate copolymer) 40: <10K Clean with surfactant, dissociating agent, acid/base
Sterilize by autoclave
HW-40: very effective with organics; phthalates; cyclodextrins; low MW nucleosides/nucleotides; peptide digests
C: 50 – 100 µm
F: 30 – 60 µm
S: 20 – 40 µm
50: 500 – 80K Compatible with organic solvents
No saccharide derivative leaching
HW-50: medium MW molecules; peptides
0.7 MPa 55: 1K – 700K High mechanical strength (5–7 bar)
High flow rate
HW-55: medium MW molecules; RNase digest; tissue plasminogen activator; RNA aggregates; glycoproteins
pH range: 2–12 65: 40K – 5000K Useful for very hydrophobic proteins
Stable bed volume
HW-65: polysaccharides; mRNA; collagenous proteins
  75: 500K – 50,000K S: high resolution, high pressure drop, low flow rate
C: low resolution, low pressure, high flow rate
HW-75: large MW molecules; amylose subfractions; plasmid DNA; replace ultracentrigation steps

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Toyopearl is a registered trademark of Tosoh Corp.

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Ultrogel®

Beaded agarose, free of fines.

Medium Fraction Ranges (proteins) Potential Applications
Matrix: A: agarose
AcA: agarose & polyacrylamide
A6: 25K – 2,400K AcA54: 5K – 70K Globular proteins
60 – 140 µm A4: 55K – 9,000K AcA44: 10K – 130K  
pH 3–10 A2: 120K – 25,000K AcA34: 20K – 350K  
    AcA22: 100K – 1,200K  

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Ultrogel is a registered trademark of IBF Biotechnics, Inc.

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