Molecules in solution, at constant pH and salt concentration, can bind to substrate, coenzyme, or inhibitor attached to agarose resin, as solution flows past resin {affinity chromatography}. After solution passes through resin, solvent at different pH and salt concentration passes through, to remove molecules from resin.
Chromatography {column chromatography} can use column with stationary solid adsorbent, such as cellulose, and moving liquid solvent.
Chromatography {gas-liquid chromatography} (GLC) can use stationary liquid phase and moving gas phase.
purpose
GLC can separate high-volatility substances, such as molecules with carbon, nitrogen, and hydrogen atoms, and determine amounts, with no decomposition. GLC needs only small sample.
process: column
Columns are polyester, silicone polymer, or diatomaceous earth, with varying mesh sizes. Mesh size determines surface area. Columns are 1 to 3 meters long.
process: solvent
Solvent saturates column material. Inert, thermally stable liquids with low vapor pressure can be solvents. Typical solvents are naphthalene or anthracene. Squalene is for non-polar molecules. Amides are for polar molecules.
process: solution
Solution starts at column end.
process: gas
Inert helium or nitrogen gas, at high pressure, is mobile phase.
process: flow
Volatile molecules in solution can separate between gas and liquid phases. More-volatile molecules spend more time in moving gas phase and move faster. High flow-rate minimizes diffusion. Heating makes molecules more volatile. Temperature is 25 C to 150 C. Lower temperature gives better resolution.
volume
Gas volumes {retention volume} elute samples.
time
Columns take time {retention time, column} to elute samples. Retention time depends on number of carbons. Bigger molecules are slower. First sample takes minimum time {dead time}.
detection
To detect sample, use Wheatstone bridge to measure conduction {thermal conductivity} (TCD).
Ionize in flame {flame ionization} (FID), if sample is solid or liquid organic but not carbonyl.
Measure decrease in electron flow {electron capture} (ECD) for halogens, oxygen, nitrogen, and sulfur compounds but not for hydrocarbons, amines, or ketones.
Chromatography {gas-solid chromatography} (GSC) can separate and determine amount, using alumina, silica, charcoal, zeolite, or polymer beads like Porapak as solid.
Liquid-liquid chromatography {high-pressure liquid chromatography} (HPLC) can use high pressure. The 100-atmosphere pressure requires special packing materials, such as Zipax or Coracil bonded beads, with porous-material coatings. Beads can have different sizes. Solvent, pH, ionic strength, and temperature affect HPLC.
purposes
HPLC separates molecules with low vapor pressure and high molecular mass or easily decomposed materials, such as nucleic acids, amino acids, bile acids, drugs, pesticides, herbicides, surfactants, and anti-oxidants. It is fast, is sensitive, and has high resolution.
Molecules or ions in solution can exchange with molecules or ions bound to polymer resin, at constant pH and salt concentration, as solution flows past resin {ion-exchange chromatography}. After solution passes through resin, solvent at different pH and salt concentration, or different solvent, passes through resin to remove bound molecules or ions.
ion types
Polystyrene or CM-cellulose sulfonated groups bind hydrogen ions or sodium ions {cation exchange}. DEAE-cellulose amine groups bind anions {anion exchange}. Anion-exchange resins, such as DEAE Sephadex, can have surface depressions for size separation.
clay
Besides polymer resins, ion-exchange chromatography can use clays, as in water softening, or sodium aluminum silicate zeolites.
chemical activity
Debye-Hückel theory relates potential-energy and chemical-potential lowering to ion solubility. Low chemical potential means solution is more random and more soluble. Ions in solution have more potential energy than uncharged molecules. Ion chemical potential depends on molecule concentration, size, and charge. Other ions go faster or slower as they pass charge. Opposite-charge counterions surround ions, making ions closer together than in random arrangements and lowering potential energy and chemical potential. Counterions shield ions and reduce effective charge, lowering potential energy and chemical potential.
chemical activity: solvent polarization
Solvent has dielectric constant and polarization. Polarization decreases attraction between ions, lowering potential energy and chemical potential. Water lowers chemical potential most, because it has highest dielectric constant.
chemical activity: factors
Lower concentration, higher temperature, higher solvent dielectric constant, lower ion charge, and larger ion size cause lower chemical potential, because potential energy is lower.
chemical activity: field
External electric field increases chemical potential. Negative voltage decreases positive-ion chemical potential, because ions have less-directed motion. Low chemical potential makes low current. Electric field can come from two different ion concentrations or two different phases. At equilibrium, potentials are equal in all phases.
Chromatography {liquid-solid chromatography} {adsorption chromatography} can separate non-polar molecules with different steric or spatial configurations. Adsorption chromatography can separate large amounts. Solid phase is silica, calcium carbonate, charcoal, or alumina, which all adsorb liquid solvents well. Eluant solvent flows past solid, carrying molecules to analyze. Bigger molecules and more polar molecules adsorb better and move slower. Second eluant can elute high-polarity molecules that stay in solid.
Liquid-liquid chromatography {paper chromatography} can separate barbiturates, antibiotics, amino acids, hormones, indoles, and ions. Paper chromatography is cheap, fast, and sensitive. Paper is stationary phase and saturates with solvent. Paper draws mobile phase along by capillary action. Fluorescent dyes stain separated molecules. Ninhydrin stains amino acids.
Chromatography {liquid-liquid chromatography} {partition chromatography} can be for polar molecules. It has high resolution, uses small batches, is more reproducible, and uses lower concentration than adsorption chromatography. Stationary phase is deactivated silica gel, diatomaceous earth, or cellulose, which absorb water. Organic solvents, such as octanol, slightly soluble in water are mobile phase. Solute has relative concentrations in octanol and water. Polarity, hydrogen bonding, and molecule size affect partition. Concentration ratio {partition coefficient} determines mobility.
Solid-liquid chromatography {thin-layer chromatography} can separate amino acids, food colorings, drugs, sugars, dyes, insecticides, ions, and salts. It is cheap, fast, and sensitive. Stationary phase is silica or alumina layer on glass plate. Solvent typically is strong acid or base.
5-Chemistry-Analytical Chemistry-Chromatography
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Date Modified: 2022.0225