HPLC (phase & phobicity) Flashcards
how does separation occur in an LC system?
relative partitioning
what makes LC and HPLC different
HPLC is when pressure is added to the system to deliver the solvent into the column
describe an HPLC system
The solvent (mobile phase) moves through a pump, the analyte is then injected into the system before entering the HPLC column (stationary phase). The compounds move through the system and the detector sends information to a computer. A fraction collector can be used to collect certain compounds.
how does pressure affect the HPLC system
separation is enhanced
components of a solvent reservoir
vent
cap
sinker frit
purpose of a sinker frit
to filter gases that shouldn’t go through the system
describe the pump
solvent delivery system
Ability to deliver up to 15,000 psi/10,200 atm
components of a HPLC system
solvent reservoir
pump
injector: manual or auto
column guard
column
detector
computer data station
fraction collector
waste collector
purpose of a column guard
stop any unfiltered material from getting into the column
what does the column containHILIC
stationary phase
most common type of detector
UV-Vis
other types of detector
refractive index
fluorescence
conductivity
electrochemical
radiochemical
evaporative light scattering detectors
mass spec
mechanism for separation in HPLC
polarity
electrical charge
size
types of liquid chromatography
normal phase
reversed phase
ion exchange
size eclusion
affinity
what types use polarity as a separation mechanism
normal phase
reverse phase
what types use electrical charge as a separation mechanism
ion exchange
what types use size as a separation mechanism
size exclusion
what mechanism/s does affinity chromatography use
charges, molecular shape/size, etc
saying for normal and reverse phase chromatography
‘like attracts like’
PRINCIPLE FOR NORMAL PHASE
molecules with similar polarity attract each other (slow down) and those with different repel (speed up), thus acheiving seperation
red blue and yellow dye is put through normal phase system. They elute yellow then red then blue. which is the most polar
blue
-it remained in the polar stationary phase for the longest
advantages to normal phase
good for very hydrophobic or hydrophilic compounds
good for water insoluble and water intolerant compounds
good for seperation of isomers
useful preparative and process scale chromatography
disadvantages to normal phase
toxicity and high cost for solvent purchase and disposal
lower BP solvents are subject to evaporation and bubble formation
silica columns can absorb water affecting reproducability of results
less widely used than reverse phase
descirbe the stationary and mobile phase of reverse phase chromtography
stationary: non-polar (C18)
mobile: polar (aqueous)
3 dyes are entered into a reverse phase system; red, blue, and yellow. Yellow elute first followed by red and then blue. Which is the most non-polar
blue
-spend the most time in the stationary phase
advantages to reverse phase
wide applicability ~75% of HPLC is RP
good for seperating a wide range of compounds
gives reproducible results
useful for preparative and process scale chromatography
disadvanatges of reverse phase
purchase and disposal of HPLC system is spenny
to high flow rates cannot be used especially for high viscosity solvents (water)
what does HILIC stand for
hydrophilic interaction chromatography
describe the stationary phase of HILIC
polar
describe the mobile phase of HILIC
polar + organic
what kind of compounds is HILIC ideal for separating
mid-high polarity and charged/hydrophilic organic compounds
example of compound HILIC can be used for
proteins (rubisco) , melamine
descibe the intial HILIC conditions
homogenous, high organic solvent mobile phase
Describe the HILIC system conditions after aqueous solvent has been added
biphasic system
half aqueous rich (stationary phase)
half aqueous depleted solvent (mobile)
what is the partitioning of analytes based on for HILIC
partitioning into solvents
dipole-dipole interactions
hydrogen bonding
describe what happens to a charged protein as it moves through
the charges align with the aqueous solvent and bond to the stationary phase slowing their movement
why is HILIC better than RP-HPLC?
better selectivity
what does HIC stand for
hydrophobic interaction chromatography
describe the stationary phase of HIC
non-polar
describe the mobile phase of HIC
high polar, high salt
role of salt in mobile phase
forces proteins to open up and expose hydrophobic areas allowing interaction with the non-polar stationary phase
what does gradually decreasing the salt content relate to elution
elution becomes based upon polarity
why is HIC useful
samples sensitive to harsh chemicals but not high salt (some anti-bodies or enzymes)
samples with high ionic strength
allows loading of large volumes of samples
summarise the stationary phase of
normal phase
reverse phase
HILIC
HIC
normal: polar
reverse phase: non-polar
HILIC: polar
HIC: non-polar
describe the mobile phase of
normal phase
reverse phase
HILIC
HIC
normal phase: non-polar
reverse: polar
HILIC: non-polar solvent, slightly aqueous
HIC: high salt, polar/ low salt +/- solvent
