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
Dead time (t_m)
Time required for the mobile phase to travel the length of the column
Retention time (t_r)
Time required for the analyte to elute from the column
Adjusted retention time (t’_r)
Retention time corrected for dead time
Retention factor (K)
A measure of the relative amount of time an analyte spends in the stationary phase
Resolution
The degree to which two peaks are separated in a chromatogram
Theoretical plates
They are a conceptual representation of a separation step. The longer the analyte is on a column, the larger the number of plates. The wider the peak, the lower the number of plates. More theoretical plates, better separation
What causes tailing, a type of asymmetrical peak shape?
When there is more than one retention mechanism for the analyte
Other causes include when a sample dilution solvent is a stronger eluent than the mobile phase, contamination of the stationary phase, and sample overload
What causes fronting, a type of asymmetrical peak shape?
Cause by bad column packing. Fixed by replacing column
Band broadening
The longer the analyte stays in the column (retention time), the broader the peak shape becomes, but total area remains constant. Therefore, peak area is better for quantitative analysis than peak height
how does plate height contribute to the peaks
More plates result in narrower peaks
Eddy Diffusion
Different substances take different paths in the column length. Leads to the diffusion of mixtures through the column
Longitudinal Diffusion
Diffusion of analyte from the concentrated middle region of the band and goes from high concentration to low concentration (center to the edges). molecules travel in the mobile phase quickly, analytes spend less time in the column
Mass transfer effect
Temperature dependent. The higher the T, the faster it is to reach eq’m and results in small mass transfer effect. The trade-off is the increase in temperature affects longitudinal diffusion, since it depends on thermodynamics (faster to diffuse)
Peak width vs. separation
Smaller peak widths are better, but the draw back is it might not be separated properly
Effect of particle size on separation efficiency
As particle size decreases, monodispesrity increases, then the various paths through the column become more uniform in distance
As particle size decreases, diffusion lengths (the distance an analyte must travel to reach the stationary phase) decrease
Totally porous particle vs superficially porous particle
Superficially porous particle provides shorter diffusion lengths and have better efficiency. Improve C term in van Deemter eq. (rate of mass transfer)
Isocratic elution
the same mobile phase composition is used throughout the separation. Not efficient in eluting things with different hydrophobicity/polarity
Gradient elution
the mobile phase composition is gradually changed during the separation. In reverse phase HPLC, the mobile phase is gradually made less polar
Temperature programming is the GC equivalent of the gradient elution in LC. T has a much greater effect in GC than LC
Gas chromatography
Still partitioning, but temperature drives analytes into the gas phase. Analyte can remain in the vapor phase, condense on the stationary phase, and dissolve in the stationary phase
GC capillary columns
long and narrow. Bonded phase on inner wall of capillary column
Split/splitless injection
insert sample into GC and often uses a syringe in narrow columns. Rapid vapourization of sample. Some bled off to waste, some enters the column (valve controls these amounts)
Flame ionization detector
Insensitive to most inorganic compounds and does not respond to fully oxidized carbons. Has very low detection limits. Good for trace analysis and is low cost. Has fast response.
Significance of Flow rate in GC
optimizing it is very important for the efficiency of GC separations than LC separations. A flow rate that’s too high or low causes broadening, which we do not want
Significance of Temperature in GC
Separates compounds into distinct identities. At an optimal temperature, the band is narrow and separate from other bands
Examples of non-polar mobile phase in liq chrom
Hexanes
Diethyl ether
ethyl acetate
dichlorochromethane
Examples of polar mobile phase in liq chrom
water
methanol
acetonitrile
propanol
