HPLC (phase & phobicity)

Question 1

how does separation occur in an LC system?

Answer 1

relative partitioning

Question 2

what makes LC and HPLC different

Answer 2

HPLC is when pressure is added to the system to deliver the solvent into the column

Question 3

describe an HPLC system

Answer 3

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.

Question 4

how does pressure affect the HPLC system

Answer 4

separation is enhanced

Question 5

components of a solvent reservoir

Answer 5

vent
cap
sinker frit

Question 6

purpose of a sinker frit

Answer 6

to filter gases that shouldn’t go through the system

Question 7

describe the pump

Answer 7

solvent delivery system
Ability to deliver up to 15,000 psi/10,200 atm

Question 8

components of a HPLC system

Answer 8

solvent reservoir
pump
injector: manual or auto
column guard
column
detector
computer data station
fraction collector
waste collector

Question 9

purpose of a column guard

Answer 9

stop any unfiltered material from getting into the column

Question 10

what does the column containHILIC

Answer 10

stationary phase

Question 11

most common type of detector

Answer 11

UV-Vis

Question 12

other types of detector

Answer 12

refractive index
fluorescence
conductivity
electrochemical
radiochemical
evaporative light scattering detectors
mass spec

Question 13

mechanism for separation in HPLC

Answer 13

polarity
electrical charge
size

Question 14

types of liquid chromatography

Answer 14

normal phase
reversed phase
ion exchange
size eclusion
affinity

Question 15

what types use polarity as a separation mechanism

Answer 15

normal phase
reverse phase

Question 16

what types use electrical charge as a separation mechanism

Answer 16

ion exchange

Question 17

what types use size as a separation mechanism

Answer 17

size exclusion

Question 18

what mechanism/s does affinity chromatography use

Answer 18

charges, molecular shape/size, etc

Question 19

saying for normal and reverse phase chromatography

Answer 19

‘like attracts like’

Question 20

PRINCIPLE FOR NORMAL PHASE

Answer 20

molecules with similar polarity attract each other (slow down) and those with different repel (speed up), thus acheiving seperation

Question 21

red blue and yellow dye is put through normal phase system. They elute yellow then red then blue. which is the most polar

Answer 21

blue
-it remained in the polar stationary phase for the longest

Question 22

advantages to normal phase

Answer 22

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

Question 23

disadvantages to normal phase

Answer 23

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

Question 24

descirbe the stationary and mobile phase of reverse phase chromtography

Answer 24

stationary: non-polar (C18)
mobile: polar (aqueous)

Question 25

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

Answer 25

blue -spend the most time in the stationary phase

Question 26

advantages to reverse phase

Answer 26

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

Question 27

disadvanatges of reverse phase

Answer 27

purchase and disposal of HPLC system is spenny to high flow rates cannot be used especially for high viscosity solvents (water)

Question 28

what does HILIC stand for

Answer 28

hydrophilic interaction chromatography

Question 29

describe the stationary phase of HILIC

Answer 29

polar

Question 30

describe the mobile phase of HILIC

Answer 30

polar + organic

Question 31

what kind of compounds is HILIC ideal for separating

Answer 31

mid-high polarity and charged/hydrophilic organic compounds

Question 32

example of compound HILIC can be used for

Answer 32

proteins (rubisco) , melamine

Question 33

descibe the intial HILIC conditions

Answer 33

homogenous, high organic solvent mobile phase

Question 34

Describe the HILIC system conditions after aqueous solvent has been added

Answer 34

biphasic system half aqueous rich (stationary phase) half aqueous depleted solvent (mobile)

Question 35

what is the partitioning of analytes based on for HILIC

Answer 35

partitioning into solvents dipole-dipole interactions hydrogen bonding

Question 36

describe what happens to a charged protein as it moves through

Answer 36

the charges align with the aqueous solvent and bond to the stationary phase slowing their movement

Question 37

why is HILIC better than RP-HPLC?

Answer 37

better selectivity

Question 38

what does HIC stand for

Answer 38

hydrophobic interaction chromatography

Question 39

describe the stationary phase of HIC

Answer 39

non-polar

Question 40

describe the mobile phase of HIC

Answer 40

high polar, high salt

Question 41

role of salt in mobile phase

Answer 41

forces proteins to open up and expose hydrophobic areas allowing interaction with the non-polar stationary phase

Question 42

what does gradually decreasing the salt content relate to elution

Answer 42

elution becomes based upon polarity

Question 43

why is HIC useful

Answer 43

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

Question 44

summarise the stationary phase of normal phase reverse phase HILIC HIC

Answer 44

normal: polar reverse phase: non-polar HILIC: polar HIC: non-polar

Question 45

describe the mobile phase of normal phase reverse phase HILIC HIC

Answer 45

normal phase: non-polar reverse: polar HILIC: non-polar solvent, slightly aqueous HIC: high salt, polar/ low salt +/- solvent