CH11_13-Chromatography Flashcards
Chromatography
a technique that separates a mixture into individual components when the mixture is carried through a stationary phase by a mobile phase
Stationary phase
-the phase that is fixed in place in chromatography column or plate;-could be solid or liquid
Mobile phase
-the phase that carries the analyte mixture through the stationary phase;-could be gas or liquid
Elution
the process in which analytes are carried through the stationary phase by the movement of the mobile phase
Differential migration
SAMPLE components migrate at different rates through the stationary phase (column)
Migration rate depends on
the strength of interaction between SAMPLE components with the stationary phase
Chromatography is useful as a tool to determine
the absence or presence of components of mixtures whose identities are known
Chromatography is more commonly used to
separate a mixture before individual components are analyzed by other techniques such as mass spectrometry
Adsorption chromatography
-a solid stationary phase and a liquid or gaseous mobile phase;-solute is adsorbed on the surface of the solid particles;-the more strongly a solute is adsorbed, the slower it travels through the column
Partition chromatography
-a liquid stationary phase is bonded to a solid surface, which is typically the inside of the silica chromatography column in gas chromatography;-solute equilibrates between the stationary liquid and the mobile phase
Ion-exchange chromatography
-anions or cations are covalently attached to the stationary phase, usually a resin;solute ions of the opposite charge are attracted to the stationary phase;-mobile phase is a liquid
Molecular exclusion chromatography
-also called size exclusion, gel filtration, or gel permeation;-separates molecules by size, with the larger solutes passing through most quickly
Affinity chromatography
-most selective;-employs specific interactions between one kind of solute molecule and a second molecule that is covalently attached to the stationary phase;-once undesired solutes have been washed from column, desired one is dislodged by changing the pH
Normal Phase chromatography
-polar stationary phase, less polar mobile phase;-least polar solute eluted first
Reversed Phase chromatography
-nonpolar stationary phase, more polar mobile phase;-least polar solute eluted last
Advantages of Reversed Phase chromatography
-water (aqueous buffer) can be used as the mobile phase (inexpensive, nontoxic, and biocompatible);-faster elution (mass transfer is faster ;-75% of all HPLC separations are performed with RP
Adsorption chromatography is ______ phase (normal or reverse); Why?
-fine, porous silica or alumina particles with large surface area; since the surface of silica (or alumina) particles are polar, adsorption chromatography is normal-phase
In HPLC, the more similar in polarity the mobile phase is to the stationary phase, the ______ the mobile phase
stronger
In HPLC, the stronger the mobile phase, the _____ the elution
faster
Diagram of typical Gas Chromatograph
Carrier gas tank -> Flow regulators -> Sample injection chamber -> Column -> Detector -> Flow meter and Data system
In a Gas Chromatograph, what makes up the Oven and Thermostat
Oven = Sample injection chamber;Thermostat = Column and Detector
Sample Introduction in GC
-Liquid samples are injected through a septum with a micro-syringe;-The sample is vaporized in the sample port;-The vaporized sample is swept onto the GC column by the mobile phase
How is the sample vaporized in the sample port in a GC
the sample port is heated around 50-C above the boiling point of the least volatile component in the sample
HPLC
using “a column packed with very small particles” and the use of a “high pressure difference across the column to drive the mobile phase” to improve the efficiency of liquid chromatography
High efficiency (in HPLC)
analytes with only slightly different properties can be separated
Modern HPLC are almost exclusively?
liquid-liquid (partition) chromatography
Diagram of HPLC
Reservoir -> Pump -> Injector -> Column -> Detector -> Data system
HPLC Column/Column packing
-usually stainless steel;-stationary phase support: porous particles of silica, alumina, or synthetic resin;particle size: 3-5 microns, 5 mostly;-a thin layer of stationary phase is coated onto the stationary phase (bonded or absorbed)
