W13 HPLC Flashcards
features of HPLC column
consists of analytical and guard columns
analytical column packings have particle sizes of 2-10 micrometer (mostly silica)
guard column increases life of analytical column by removing particulate matter and contaminants from solvents
guard columns contain the same particulate packing material and stationary phase as analytical column but shorter
stationary phase
highly pure, spherical particles
usually porous and solvent permeable > have very large surface area
covalently attached to polymer
why does HPLC needs a packed column
small solid particles > increase surface area to volume ratio > more binding sites for solutes > can equilibrate faster between 2 phases > decrease in mass transfer resistance > increase resolution
why is it difficult to combine HPLC with MS
ionisation issues: GC uses gases that are easily ionised in MS but HPLC uses liquid mobile phase > introducing this liquid into MS can overwhelm the system
high vacuum requirement: MS requires high vacuum (no air) > but large volume in HPLC hard to evaporate quickly enough to maintain this vacuum
non-volatile compounds: HPLC designed to separate non volatile and polar compounds that do not vaporise easily > but MS optimised for gas phase ions
high flow rates: HPLC rates higher than those in GC > increased liquid volume must be managed by flow splitting or using very low flow rates to avoid MS overload
why does using smaller particles lead to improved resolution
more uniform filling > smaller A (multiple flowpaths)
distance solute must diffuse through is smaller > lower C (mass transfer resistance)
disadvantage of small particles
increased resistance to solvent flow > may be hard to maintain desired flow rate
normal phase HPLC
stationary phase is polar, mobile phase is non polar
least polar component is eluted first
increasing polarity of mobile phase > compete with polar component > polar component interact less with stationary phase > decrease elution time
reverse phase HPLC
stationary phase is non polar, mobile phase is polar
most polar component is eluted first
increasing polarity of mobile phase > non polar component elution time increases
elution order
methanol > acetone > toluene > hexadecane
isocratic elution
performed with single solvent or constant solvent mixture
usually when analyses have similar interactions with stationary phase and elute at similar times
gradient elution
the continuous change of solvent composition to increase fluent strength which is required to elute more strongly retained solutes
mobile phase reservoirs and pumping
dissolved gases (N2 and O2) in liquids produce bubbles in column > band spreading
dissolved gases removed by bubbling inert gas (He) which has low solubility in mobile gas
what is loop injector
device used in chromatography to inject precise amounts of sample onto column
sample is loaded into a short section of tubing and injected onto column by redirecting mobile phase through the loop
