W8 HPLC and FPLC Flashcards

1
Q

basic components of liquid chromatography

A

pump > sample loop > column > detector > fraction collector and waste collector

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2
Q

parameters affecting separation process in LC

A

pressure, flow rate, capacity and target molecule

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3
Q

how does size exclusion chromatography work

A

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

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4
Q

what is frictional resistance

A

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

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5
Q

definition of hydrophobicity

A

the association of non polar groups or molecules in an aqueous environment which arises from the tendency of water to exclude non polar molecules

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6
Q

what is resolution

A

a quantitative measure of how well two elution peaks can be differentiated in a chromatographic separation

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7
Q

important parameters of purification column

A

capacity: volume (usually no more than 5% of column volume) and amount

column volume

void volume

flow rate

pressure limit

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8
Q

what are protein tags

A

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

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9
Q

how to improve resolution for ion exchange chromatography

A

decrease sample load

decrease flow rate

use shallower elution gradient

use smaller resin particle size

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10
Q

difference between normal phase HPLC and reverse phase HPLC

A

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

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11
Q

factors affecting elution efficiency

A

hydrophobicity/polarity of mobile phase

column material

loading capacity

impurities

slope of gradient

flow rate

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12
Q

difference between FPLC and HPLC

A

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

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