Term
| History of Chromatography |
|
Definition
1900 – Russian-Italian Botanist Mikhail Tsvet was studying chlorophyll
Developed a liquid-adsorption column containing calcium carbonate to separate plant pigments
Called it “chromatography” for “color writing”, color also referring to his last name |
|
|
Term
|
Definition
Separation of analyte
Immobilized phase (stationary phase)
Mobile phase (effluent) |
|
|
Term
|
Definition
Applied to volatile organic compounds
The mobile phase is a gas
The stationary phase a solid adsorbent |
|
|
Term
| Liquid Chromatography (LC) |
|
Definition
Used to separate metal ions and organic compounds in solution
The mobile phase is a solvent and the stationary phase is a solid, gel, or resin |
|
|
Term
| Thin Layer Chromatography (TLC) |
|
Definition
A simple and rapid method to monitor the extent of a reaction or to check the purity of organic compounds
The mobile phase is a solvent and the stationary phase is a solid adsorbent (such as paper, cellulose, or silica) |
|
|
Term
| High Performance Liquid Chromatography (HPLC) |
|
Definition
1970s
A variation of LC that utilizes high pressure pumps (up to 100 MPa) to increase the efficiency of the separation (keep constant flow)
Ability to separate, identify and quantitate the compounds that are present in any sample that can be dissolved in a liquid
Can separate ppt (parts per trillion)
Pharmaceuticals, food, cosmetics, environmental samples, forensic samples, industrial chemicals, nucleotides, peptides
Resins/columns/samples must be able to withstand high pressures |
|
|
Term
| Size Exclusion Chromatography (SEC) |
|
Definition
Starch columns were used to analyze amino acids and proteins in 1940s
In 1956 by Grant Lathe and Colin Ruthven (London) showed multiple proteins could be separated by size on a starch column
Arne Tiselius (who worked under Theodor Svedberg) (Sweden) discovered that bacteria on beets were producing dextran (Tiselius’s student Stellan Hjertén developed acrylamide)
Jerker Porath and Per Flodin (Sweden) cross-linked dextran and showed proteins could be separated by size
SEC – also called gel filtration or gel permeation chromatography
The mobile phase is a solvent and the stationary phase is a packing of porous particles |
|
|
Term
| Size Exclusion (Gel Filtration) Chromatography |
|
Definition
SEC is a mild method for purifying nucleic acids, sugars, and proteins:
Separating molecules groups based on size (MW)
Desalting (buffer exchange)
Removing ammonium sulfate after a precipitation
Reproducible
The sample is applied in a
narrow band at the top of
the column and then
washed through the column
across the mobile phase
Disadvantage: band
broadening (inject <10 ml) |
|
|
Term
|
Definition
The amount of cross-linking determines the average pore size of a gel
Different degrees of cross-linking give different pore sizes and different molecular sieving or separation ranges
Three types of polymers
Dextran (Sephadex) beads,
20-300 microns
Polyacrylamide
Agarose |
|
|
Term
|
Definition
Synthesized from sucrose by bacteria
Leuconostoc mesenteroides B-512F
Decreases thrombosis
Lubrication in eye drops
Blood replacement
Increase sugar levels
Dextran is cross-linked to various extents by reaction with epichlorohydrin to give gel beads with different pore sizes
Useful for proteins |
|
|
Term
| Size Exclusion Chromatography |
|
Definition
Separation based on size and shape
Contains porous beads typically 100 mm in diameter
Beads have an “exclusion size”, in the QBM lab we use Sephadex G-75 beads
Total liquid volume >95% of the packed column |
|
|
Term
| Preparative Applications of SEC |
|
Definition
Compatible with physiological conditions (pH, salts) and can be used with most protein samples
Large sample protein separation
Proteins (or other macromolecules) of different molecular weight
Monomers from dimers, trimers, tetramers, etc.
Separate monomers from aggregated proteins or degraded products, which often form during protein purification
SEC is often incorporated as a final polishing step to remove aggregates and act as buffer exchange mechanism into the final solution |
|
|
Term
|
Definition
|
|
Term
|
Definition
Bead size is an average
Pore size is an average
(50-1000 angstroms)
Other factors: flow rate,
viscosity
Separate molecules ~2X
difference in Mw |
|
|
Term
|
Definition
Stokes radius is the effective radius a molecule has as it tumbles rapidly in solution (i.e. the radius of a hard sphere that diffuses at the same rate as the molecule)
A long extended molecule has a larger stokes radius than a compact molecule of the same molecular mass |
|
|
Term
| Analytical Applications of SEC |
|
Definition
SEC columns are used analytically for studying
Protein purity
Protein folding
Protein-protein interactions
Quaternary structure
Mw with a calibration (standard) curve |
|
|
Term
| Chromatography Components |
|
Definition
Chromatography column
Gel matrix
Buffers
Gradient mixer
Peristaltic pump
Conductivity meter
UV detector
Fraction collector |
|
|
Term
|
Definition
SEC
Prepacked
Dry powder
Column length affects resolution
IEX (anion, cation)
Nickel
GST
Maltose
Columns have a maximum load, and longer/larger columns will hold more protein |
|
|
Term
|
Definition
Increase salt or other eluting molecule
Attach the gradient mixer to the column
Open the valves
Can be performed by the chromatography machines in lab |
|
|
Term
| Collecting the Column Fractions |
|
Definition
Collect the column effluent using an automated fraction collector
Measure the A280 of each fraction
Column fractions can be collected by time or by drop number (preferred to prevent variations) |
|
|
Term
| Detecting the Proteins in the Column Fractions |
|
Definition
A280 of the individual chromatographic fractions (chromatogram)
Bradford/DC protein assay
The biological activity of aliquots from the individual chromatographic fractions can be assayed (CDNB assay)
SDS-PAGE (size)
Western blot (antibodies) |
|
|
Term
|
Definition
Protein did not elute
Charge interactions with column? Run in salt buffer
Hydrophobic interactions with column?
Air bubbles in gel
Matrix was not degassed before pouring gel
Column moved from cold to warm location
Cracks in gel
Column ran dry
No column flow, and increased pressure
Clogged, tubing is blocked, valves not open
Reduced column flow (or increased pressure)
Insoluble particles in sample, and plugging of column by fine particles (clean with NaOH)
Compressed column bed
Microbial growth (store in 20% absolute EtOH or 1% sodium azide) |
|
|
