W8 HPLC and FPLC Flashcards
basic components of liquid chromatography
pump: to pump the mobile phase containing sample through the column to interact with stationary phase
sample loop: to connect the sample ejector to the rest of the chromatographic system
column: contains stationary phase particles to separate the analyte from rest of sample
detector to detector analyte
fraction and waste collector
parameters affecting separation process in LC
pressure, flow rate, capacity and target molecule
definition of hydrodynamic radius
the effective radius of a particle as it moves through a solution > take into account its size and interaction of solvent
protein denatured > non structured > more surface area exposed > hydrodynamic radius increases
how does size exclusion chromatography work
large molecules cannot enter the pore > flow around the beads and interact less than small molecules > travel faster through the column
smaller molecules travel through column at slower rate
what is frictional resistance
a force of opposite direction to the flow velocity that occurs when object move in a flow
F = - frictional coefficient x flow velocity
frictional coefficient is proportional to viscosity of solution and hydrodynamic radius of the solute
definition of hydrophobicity
the association of non polar groups or molecules in an aqueous environment which arises from the tendency of water to exclude non polar molecules
what is resolution
a quantitative measure of how well two elution peaks can be differentiated in a chromatographic separation
important parameters of purification column
capacity: volume (usually no more than 5% of column volume) and amount
column volume
void volume
flow rate
pressure limit
what are protein tags
a short peptide with unique a.a sequence designed to fuse at N or C terminus
can be cleavable
tag can interact with specific groups on stationary phase at high affinity
how to improve resolution for ion exchange chromatography
decrease sample load: less crowding > more time to interact with resin and separate properly
decrease flow rate: more time for proteins to bind and separate efficiently
use shallower elution gradient: separates proteins with similar binding strengths more effectively
use smaller resin particle size: more surface area for interactions between protein and column > sharper separation of proteins with similar properties
difference between normal phase HPLC and reverse phase HPLC
normal: solid phase is more polar than mobile phase > solute with higher polarity will interact with column ; usually used for small polar molecules
reverse: solid phase is less polar > component with lower polarity will interact with column ; usually used for large molecules like peptide and protein
factors affecting elution efficiency
hydrophobicity/polarity of mobile phase
column material
loading capacity
impurities
slope of gradient
flow rate
difference between FPLC and HPLC
different target: macromolecule (protein, peptide, protein complex) for FPLC and small molecule (peptide, chemical compound) for HPLC
operation pressure: 40 Bar/4 MPa for FPLC and 1500 Bar/150 MPa for HPLC
mobile phase: aqueous buffer for FPLC and solvent for HPLC
column material: agarose/polymer for FPLC and silica beads for HPLC
properties of FPLC
operates at pressure of 40bar and flow rate of 0.5-1 ml/min
uses aqueous buffers like Tris, PBS, MES or HEPES
column made of agarose or polymer beads
uses UV detector to monitor proteins
ideal for proteins and peptides in their natural (non-denatured) state
factors that determine purification efficiency for gel filtration/size exclusion
loading capacity, separation capacity, total column volume
difference between target protein and impurities
inherent features of protein: structure and stability etc
