Unit 5 - Intro to Separation Science and Liquid Chromatography Flashcards
Separation science
separate and isolate components of the mixture, then determine amount of each analyte
Liquid-liquid extraction
Separatory funnel separates two immiscible liquids and the analytes are separated between two phases. The partition coefficient depends on solvent, pH, ionic strength, and temp
Solid-liquid extraction
Coated with hydrophobic/hydrophilic extraction phase. If coating is nonpolar, it’ll help extract nonpolar compounds from the solution. Same applies to polar
Elution chromatography
Column has a stationary phase and mobile phase and the analyte partitions depending on its affinity towards the stationary and mobile phase. Depends on polarity
Multistep phase separation
use counter-current extraction. Put sample in and certain molecules will prefer the mobile or immobile phase and move. When eq’m is achieved, repeat process until the molecules are separated and is pure
Adsorption chromatography
Some molecules are attracted to the surface of the chromatography and are held back. Too much of these molecules will cause surface saturation and it won’t adsorb on the surface. Partition coefficient goes down because of overload
NOTE: the stationary phase is a solid and the mobile phase is usually liquid or gas
Solid adsorption media examples
silica (SiO2) - polar, slightly acidic
alumina (Al2O3) - polar, neutral or slightly basic
Thin layer chromatography (TLC)
Quick and simple; low cost
A mixture is spotted on silica or alumina (stationary phase) and the bottom plate is immersed in solvent and travels up the plate by capillary action (mobile phase) and allows the mixture to separate into spots or bands
Column chromatography (purifies reaction products)
Column contains silica gel or alumina and load mixture on top of the column. Elute with organic solvent as mobile phase and the bands for each component separate out, which is then collected
(think of NH4+ experiment in lab)
High Performance Liquid Chromatography (HPLC) and Ultra Performance Liquid Chromatography (UPLC)
Stationary phases are made up of small solid particles. As particle size decreases, separation efficiency increases because the column is more densely packed. Drawback is the very high back-pressure, where gravity is insufficient to move the mobile phase through the column and pumps are required
Analytical and Preparative columns
Analytical columns are used for trace analysis and detection. For particles smaller than 10 micrometers
Preparative columns are used for purification of synthesized compounds. Usually larger than analytical columns
Bonded stationary phases - silica particles
The silica particles are packed inside columns. These particles are coated with functional groups that modify their polarity
Partition Chromatography (absorption)
There are two liquids that separate the solutes. One liquid is the stationary phase, and the other is the mobile phase, and the solutes prefer one or the other due to their partition coefficients. The solute is dissolved in the liquid phase that is coated on the surface of solid support
Normal and Reverse phase HPLC
Normal phase HPLC separates polar molecules and is generally more stable than reverse phase. Its stationary phase is polar: silica, amino, cyano. Reverse phase separates non-polar molecules and is more commonly used. Its stationary phase it nonpolar: C18, C8, phenyl. The elution of analytes are inverted between normal and reverse phase HPLC
What does sparging mean in HPLC instrumentation?
It is when helium is bubbled through the solvent reservoirs to sweep out dissolved air since helium is insoluble in most solvents
Significance of the flow cell in HPLC UV-VIS detectors
They maximize sensitivity by increasing the path length term, b, in the Beer-Lambert Law, A=Ebc, because of its Z-shape. Sensitivity is determined by Eb
Significance of retention time in HPLC chromatogram
analytes can be differentiated and identified on the bases of their retention time. The size of the peaks indicates something about the quantity of the corresponding analyte