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FOSC301 > HPLC (phase & phobicity) > Flashcards

HPLC (phase & phobicity) Flashcards

1
Q

how does separation occur in an LC system?

A

relative partitioning

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2
Q

what makes LC and HPLC different

A

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

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3
Q

describe an HPLC system

A

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.

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4
Q

how does pressure affect the HPLC system

A

separation is enhanced

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5
Q

components of a solvent reservoir

A

vent
cap
sinker frit

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6
Q

purpose of a sinker frit

A

to filter gases that shouldn’t go through the system

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7
Q

describe the pump

A

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

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8
Q

components of a HPLC system

A

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

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9
Q

purpose of a column guard

A

stop any unfiltered material from getting into the column

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10
Q

what does the column containHILIC

A

stationary phase

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11
Q

most common type of detector

A

UV-Vis

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12
Q

other types of detector

A

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

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13
Q

mechanism for separation in HPLC

A

polarity
electrical charge
size

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14
Q

types of liquid chromatography

A

normal phase
reversed phase
ion exchange
size eclusion
affinity

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15
Q

what types use polarity as a separation mechanism

A

normal phase
reverse phase

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16
Q

what types use electrical charge as a separation mechanism

A

ion exchange

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17
Q

what types use size as a separation mechanism

A

size exclusion

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18
Q

what mechanism/s does affinity chromatography use

A

charges, molecular shape/size, etc

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19
Q

saying for normal and reverse phase chromatography

A

‘like attracts like’

20
Q

PRINCIPLE FOR NORMAL PHASE

A

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

21
Q

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

A

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

22
Q

advantages to normal phase

A

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

23
Q

disadvantages to normal phase

A

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

24
Q

descirbe the stationary and mobile phase of reverse phase chromtography

A

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

25
Q

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

A

blue
-spend the most time in the stationary phase

26
Q

advantages to reverse phase

A

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

27
Q

disadvanatges of reverse phase

A

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

28
Q

what does HILIC stand for

A

hydrophilic interaction chromatography

29
Q

describe the stationary phase of HILIC

A

polar

30
Q

describe the mobile phase of HILIC

A

polar + organic

31
Q

what kind of compounds is HILIC ideal for separating

A

mid-high polarity and charged/hydrophilic organic compounds

32
Q

example of compound HILIC can be used for

A

proteins (rubisco) , melamine

33
Q

descibe the intial HILIC conditions

A

homogenous, high organic solvent mobile phase

34
Q

Describe the HILIC system conditions after aqueous solvent has been added

A

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

35
Q

what is the partitioning of analytes based on for HILIC

A

partitioning into solvents
dipole-dipole interactions
hydrogen bonding

36
Q

describe what happens to a charged protein as it moves through

A

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

37
Q

why is HILIC better than RP-HPLC?

A

better selectivity

38
Q

what does HIC stand for

A

hydrophobic interaction chromatography

39
Q

describe the stationary phase of HIC

A

non-polar

40
Q

describe the mobile phase of HIC

A

high polar, high salt

41
Q

role of salt in mobile phase

A

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

42
Q

what does gradually decreasing the salt content relate to elution

A

elution becomes based upon polarity

43
Q

why is HIC useful

A

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

44
Q

summarise the stationary phase of
normal phase
reverse phase
HILIC
HIC

A

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

45
Q

describe the mobile phase of
normal phase
reverse phase
HILIC
HIC

A

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

46
Q
A

FOSC301 (17 decks)

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