Exam 3 Flashcards
What is chromatography
*general term applied to wide variety of separation techniques based on patitioning of sample (solute) between moving (mobile) phase and a fixed (stantionary) phase
*relative interaction of the solute with these two phases is described by the partition coefficient
what is the mobile phase (ex.)
gas (GC), liquid (LC), or supercritical fluid (SFC)
what is the stationary phase
liquid or solid
what determined the elution time
interaction of the solute with the stationary phase
Thin-Layer Chromotography
*separation based on polarities
*TLC plates with thin layer of stationary phase
*sorbents used: silica gel, alumina, diatomaceous earth, cellulose
*TLC put into solvent (mobile phase)
*subst. with polar prop. will interact strongly with stationary phase (move slower)
*subst. with nonpolar prop. will interact strongly with mobile phase (move up & faster)
How can separation occur in TLC
*adsorptive
*partition (normal/reversed phase)
*ion exchange
*size-exclusion
Advantages & applications to TLC
ADVANTAGES:
*better resolution, faster, more reproducible, easier to handle
APPLICATIONS:
*screen corn/peanuts for mycotoxins before processing, lipids, carbs, vitamins, amino acids, pigments
Visualization/ Quantitation of TLC
VISUALIZATION:
*colorimetric, fluorescence, UV light
QUANTITATIVE:
*scraping off the zone, eluting the compound and then analyze the resulting solution
Column Chromotography (basics)
*separation to isolate/separate solutions
*mobile phase = liquid
*stationary phase = solid or liquid supported by inert solid
*stationary phase is packed in column and equilibrated with mobile phase
*mobile phase moves through column by a pump
*components will separate as they move down column
What are the essential components of a column chromotagraphy system
*magnetic stirrer
*peristatic pump
*column
*spectrophotometer
*fraction collector
Adsorption
*solute is adsorbed on surface of stationary phase (silica, alumina)
*elution order of solutes depends on relative polarities (polar = last, nonpolar = first)
*Van der waals, electrostatic, H bonding, hydrophobic interactions
*uses: separates aromatic or aliphatic nonpolar compounds, base primarily on type of # of functional groups present
External Standards
standard solutions are chromatographed and peak area (or height or mass) are plotted vs. concentration on standard curve
Internal Standards
*amount of each component in sample is determined by comparing area (or height or mass) of component peak to that of the internal standard peak
*need to run set of standards at varying concentrations, with each solution containing constant amount of internal standard
Partition Chromatography
*liquid-liquid chromatography
*hydrophobic remains in organic phase
*hydrophilic remains in aqueous phase
*column with particle of solid support coated with liquid stationary phase
*pour mixture into column and mixture dissolves into stationary phase
*mobile phase will be poured in and one comp. will interact strongly with mobile while other interacts with stationary phase
*collected as fractions
Normal Partition Chromatography
*polar stationary phase vs. nonpolar mobile phase (nonpolar solutes elute first)
*common stationary phases: silica, organic moieties w/ cyano and amino funct. groups
*uses: separate and quantitate polar hydrophilic substances, ex. carbohydrates, water-soluble plant pigments
Reversed Partition Chromatography
*nonpolar stationary phase vs. polar mobile phase
*polar solutes first to elute
*common stationary phase: alkyl hydrocarbons
*uses: separate and quantitate lipophilic compounds ex. lipids and fat soluble pigments, separate and quantitate polyphenols
Ion-Exchange Chromatography
*solid-liquid chromatogrpahy
*based on attraction between opposing charges
*stationary phase contains fixed funcitonal groups with either positive or negative charge
*cation exchnager (has neg. charge)
*anion exchanger (has pos charge)
*elution: change mobile-phase pH or inc ionic strength so solute no longer binds
*uses: amino acids, sugars (HPAEC), alkaloids, proteins, drugs, fattya cids, acids of fruit
Size-Exclusion Chromatography
*solid-liquid chromatography
*molecules are separated based on size
*stationary phase is porous
*molecules too large to enter the pores travel with the mobile phase in the interstitial phase at elute first
*solutes of low molecular weight get slowed down by entering the pores
Affinity Chromatography
*solid-liquid chromatography
*isolates biomolecule from sample
*separation based on reversible interaction between a solute and an immobilized ligand (antibodies, enzyme inhbitiors, lectins)
*pour mobile phase into column w/stationary phase
*biomolecule will attach to ligand
*everything else will be washed away
*then another solvent will be poured in that competes with biomolecule to bind to ligand
*biomolecule will be separated/isolated
*uses: separate and purify enzymes or enzyme inhibitors, separate and purify glycoproteins
what is special about HPLC
*it can separate parts of a compound, tell you how much of each compound is in a mixture, and help identify what each compound is