Chromatography Flashcards
What do the terms Stationary Phase and Mobile Phase refer to?
Stationary Phase: Where the separation happens.
Mobile Phase: move the sample through the stationary phase - may also interact with the sample.
What is a chromatogram?
The graph of detection versus the retention time - peak area.
Define Eluent, Elution, Eluate and Elution Order
Eluent: The mobile phase
Elution: The analyte coming off the column
Eluate: The fluid in the column.
Elution Order: The order in which things come off the column.
what is the relative polarity of the stationary phase compared to the mobile phase in reverse phase?
Stationary: non-polar or weakly polar
Mobile: more polar
What is the relative polarity of the stationary phase compared to the mobile phase in normal phase?
Stationary: polar - solvent less polar
Mobile: a more polar solvent
what is the basis for normal phase and reverse phase separations?
Normal: compounds are attracted to, or not, the stationary phase or mobile phase based on the polarity.
Stationary: rely on differences in the hydrophobicity / polarity for separations - most common mode of HPLC.
What is ion exchange chromatography?
- Analytes are removed by flushing with mobile phase OR by changing the composition of the mobile phase.
- Stationary phase: anions or cations are covalently attached to the stationary phase - usually resin.
- Charge of the resin-bound ions balanced by weakly bound counterions such as H+ (Cl-)
- Mobile phase: liquid
- Ions are separated based on differences in the strength with which they bind with resin-bound.
What is size exclusion chromatography?
stationary phase: porous gel
mobile phase: liquid or gas
separates molecules by size, with larger solutes passing through most quickly.
- too large molecules: enter pores stream past the porous gel coating or particles and exit the column rapidly
- small molecules: enter the pores, sheltered from mobile phase flowing around gel particles (elute more slowly)
how does size exclusion chromatography differ from gel permeation chromatography?
Size exclusion: uses water traditionally.
Gel permeation: uses organic solvents traditionally.
what is the simple schematic of an HPLC
solvent (mobile phase) –> pump –> injector (autosampler) –> HPLC column (packing material) –> detector –> chromatogram
what is the difference between binary and quarternary pumping?
Binary: two pumps - can only mix 2 solvents at a time.
Quarternary: one pump - can mix up to 4 solvents at a time (proportioning valve)
what is an isocratic method? gradient?
Isocratic: can stay unchanged during entire run - simpler
Gradient: can change composition with time - can help to analyze a sample with very different compounds.
what are the two types of injectors for an HPLC
Manual Injectors: introduce the sample to the instrument.
Autosampler: can accurately partially fill a loop (more volume options)
what are the criteria for a carrier gas?
it must be pure and non reactive - to eliminate ghost peaks/contamination.
what are common carrier gases?
He, H2, N2, Ar
what is the role of a septa in the injector?
what happens when it fails?
Septa: acts as a barrier between the GC and the outside world.
Fails: loss of analyte and contamination.
what temperature range relative to the analyte do we aim for? why?
heated to >50 C above the bp of the least volatile component of the sample (if known) - to ensure quick vaporization.
What phases can we inject into GC?
Gas
why do we want fast injection?
means the sample will enter the column as a tight band.
if too slow or too big of a sample, it will result in band spread and decreased resolution.
what are the two types of columns available?
compare them.
Packed column: glass or stainless steel tubes packed with a solid material that has the liquid adsorbed to the surface.
Capillary column: open tubes - fused silica with an adsorbed liquid layer (stationary) on the inner surface (outer surface coated in polymer to reduce fragility)
What must the analyte have or do in relation to the stationary phase for retention?
analyte must have some compatibility / solubility with the stationary phase.
like dissolves like (polarity)
What is an FID? what does it detect? what are the advantages?
“Flame Ionization Detector”
The current is proportional to the amount of carbon entering the flame, which is proportional to the total analyte amount.
Advantages: high sensitivity
low noise
rugged and easy to use
what other detectors exist?
Mass Spectrometry
Thermal Conductivity
